This article was downloaded by:[EBSCOHost EJS Content Distribution]On: 26 March 2008Access Details: [subscription number 768320842]Publisher: Informa HealthcareInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Current Eye ResearchPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713618400

Involvement of Purinergic P2 Receptors in ExperimentalRetinal NeovascularizationSylvia Sarman a; Jorge Mancini b; Ingeborg van der Ploeg a; J. Oscar Croxatto c;Anders Kvanta a; Juan E. Gallo ba Department of Clinical Neuroscience, St. Erik's Eye Hospital, Karolinska Institutet,Stockholm, Swedenb Department of Ophthalmology, Hospital Universitario Austral and Facultad deCiencias Biomedicas, Universidad Austral, Pilar, Argentinac Department of Ocular Pathology, Fundacion Oftalmologica Argentina "JorgeMalbran,", Buenos Aires, Argentina

First Published on: 01 March 2008To cite this Article: Sarman, Sylvia, Mancini, Jorge, van der Ploeg, Ingeborg,

Croxatto, J. Oscar, Kvanta, Anders and Gallo, Juan E. (2008) 'Involvement of Purinergic P2 Receptors in ExperimentalRetinal Neovascularization', Current Eye Research, 33:3, 285 - 291To link to this article: DOI: 10.1080/02713680701885470URL: http://dx.doi.org/10.1080/02713680701885470

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction,re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expresslyforbidden.

The publisher does not give any warranty express or implied or make any representation that the contents will becomplete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should beindependently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with orarising out of the use of this material.

Dow

nloa

ded

By:

[EB

SC

OH

ost E

JS C

onte

nt D

istri

butio

n] A

t: 13

:09

26 M

arch

200

8

Current Eye Research, 33:285–291, 2008Copyright ©c Informa Healthcare USA, Inc.ISSN: 0271-3683 print / 1460-2202 onlineDOI: 10.1080/02713680701885470

Involvement of Purinergic P2 Receptors inExperimental Retinal Neovascularization

Sylvia SarmanDepartment of ClinicalNeuroscience, St. Erik’s EyeHospital, Karolinska Institutet,Stockholm, Sweden

Jorge ManciniDepartment of Ophthalmology,Hospital Universitario Austral andFacultad de Ciencias Biomedicas,Universidad Austral,Pilar, Argentina

Ingeborg van der PloegDepartment of ClinicalNeuroscience, St. Erik’s EyeHospital, Karolinska Institutet,Stockholm, Sweden

J.Oscar CroxattoDepartment of Ocular Pathology,Fundacion OftalmologicaArgentina ”Jorge Malbran,”Buenos Aires, Argentina

Anders KvantaDepartment of ClinicalNeuroscience, St. Erik’s EyeHospital, Karolinska Institutet,Stockholm, Sweden

Juan E. GalloDepartment of Ophthalmology,Hospital Universitario Austral andFacultad de Ciencias Biomedicas,Universidad Austral,Pilar, Argentina

ABSTRACT Purpose: The present study was aimed to investigate the expres-sion of purinergic P2 receptors in oxygen-induced retinal neovascularization.Methods: Immunohistochemistry was used to study the expression of purinergicP2Y2 and P2X2 receptors in the neonatal mouse retina during normal vasculardevelopment and after oxygen-induced retinopathy (OIR). The effect of theP2 antagonists, suramin and PPADS, on the extent of oxygen-induced retinalneovascularization was analyzed. Results: In normal mice, the expression ofP2Y2 receptors was weak throughout the retina, whereas P2X2 receptor expres-sion was detected in the outer plexiform layer. In mice treated with oxygen,P2Y2 expression was detected in the ganglion and in the nerve fiber layers,whereas P2X2 expression was found in the inner and outer plexiform layers.Oxygen-induced preretinal neovascularization was strongly inhibited by the P2antagonists, suramin ( p < 0.05) and PPADS ( p < 0.05), and this was accompa-nied by a down-regulation of P2X2 receptor expression in the inner plexiformlayer in suramin-treated mice. Conclusions: The data suggest that purinergic P2receptors are involved in neovascularization associated with OIR.

KEYWORDS angiogenesis; hypoxia; P2 receptors; retina; ROP

INTRODUCTIONThe cell surface receptors for extracellular purine and pyrimidine nucleotides

are called P2 receptors. The P2 receptors are divided into two groups: P2X andP2Y receptors. P2X receptors are cation-selective channels with almost equalpermeability to Na+, K+, and significant permeability to Ca2+. They gate ex-tracellular cations in response to ATP. P2X receptors are widely expressed in thecentral nervous system and in the body periphery, where they play importantroles in different processes, including muscle contraction, modulation of thecardiovascular and respiratory systems, and transmitter release. P2Y receptorsare G-protein coupled receptors that are divided into two subfamilies. The firstfamily is composed of P2Y receptors 1, 2, 4, 6, and 11 that predominantly cou-ple to Gq, thereby activating phospholipase C, which results in mobilization ofintracellular Ca2+. The second family consists of P2Y receptors 12, 13, and 14that are Gi-coupled and inhibit adenylyl cyclase and regulate ion channels.TheP2Y receptors account for broad and varied physiological responses such asplatelet aggregation, granulocyte differentiation, and regulation of vascular

Received 13 September 2007Accepted 18 December 2007

Correspondence: Sylvia Sarman, M.D., St.Erik’s Eye Hospital, Polhemsgatan 50,SE-112 82 Stockholm, Sweden. E-mail:sylvia.sarman@sankterik.se

285

Dow

nloa

ded

By:

[EB

SC

OH

ost E

JS C

onte

nt D

istri

butio

n] A

t: 13

:09

26 M

arch

200

8

tone.1–4 Recent studies have suggested a role forP2 receptors in retinal development5,6 and in reti-nal disease, including retinal detachment,7 prolifera-tive vitreoretinopathy,8 diabetic retinopathy,9–11 andretinal degeneration.12

A direct role for extracellular ATP and P2 receptorsin ischemic stress has been reported in various cellularsystems, and changes in ATP levels have been shownin ischemia of the CNS including the retina.8,13–15 Inmicroglia from neonatal P0 rat retina, under hypoxicconditions, it was found that the mitotic activation in-duced by hypoxia coincided with P2Y2 expression, andthat the mitotic activity was suppressed by suramin, aP2 receptor antagonist.16 P2Y2 receptors have also beenshown to be involved in the regulation of the mitoticactivity in astrocytes5 and neuroepithelial cells in theearly embryonic chick retina.6

Pathologic neovascularization is a complex multistepprocess termed angiogenesis,17 which involves remod-eling of the existing vasculature through stimulation ofangiogenic growth factor receptors on vascular endothe-lial cells, proteolytic breakdown of the endothelial basalmembrane, endothelial cell proliferation and migra-tion, degradation of the surrounding extracellular ma-trix (ECM), vessel stabilization, recruitment of support-ing cells (i.e., pericytes), and closure of the newly formedarteriovenous loops.18–20 In most cases, the drivingforce behind the angiogenic response is tissue ischemia,as seen in proliferative diabetic retinopathy or retinopa-thy of prematurity. The expression and role of P2 recep-tors during retinal neovascularization is not known.

Our present study was aimed to investigatewhether P2 receptors were expressed in oxygen-inducedretinopathy (OIR), a model for retinal neovasculariza-tion, and if the induced retinopathy was affected by theP2 receptor antagonists, suramin and PPADS.

MATERIALS AND METHODSAnimals

C57BL/6J mice were obtained from Charles River,Germany, and from BK, Sollentuna, Sweden. Both fe-male and male mice were used in the experiments.

ImmunohistochemistryFive eyes (5 animals) from either hyperoxia- or non-

hyperoxia-treated mice were enucleated and fixed for48 hr at 4◦C in paraformaldehyde (Sigma-Aldrich, St

Louis, MO, USA). Afterward, eyes were immersedfor cryoprotection in graded sucrose solution (5%overnight, 10%, 15%, and 20%) and interlockedwith resin. Fourteen micron sections were obtained(Shandon AS325 Retraction Microtome, Thermo Sci-entific, Waltman, MA, USA) and fixed on polylysine-treated glass slides. After overnight incubation inprimary antibody (P2X1 1:1000, P2X2 1:1000, P2X31:1000, and P2Y2 1:800) (Neuromics, Minneapolis,MN, USA) at 4◦C, sections were treated with biotiny-lated secondary antibodies followed by an avidin per-oxidase complex step (Vectastin Elite ABC, VectorLaboratories, Burlingame, CA, USA). Finally, a colorreaction was obtained using 3,3′-diaminobenzidine(DAB)/nickel-enhanced solution for staining (Sigma-Aldrich, St Louis, MO, USA). At least 6 sectionsper retina were analyzed. For immunofluorescence,the sections were labeled with lissamine rhodamine-conjugated goat anti-rabbit Ig-G or with fluorescein-5-isothiocyanate-conjugated goat anti-guinea pig Ig-G(Jackson Immunoresearch Laboratories, West Grove,PA, USA). At least 6 sections per retina were analyzed.Visualization was done using a Nikon FluorescenceEclipse Microscope (Tokyo, Japan), and photographswere taken with a Nikon DN 100 Digital Camera(Tokyo, Japan).

Pathologic Retinal AngiogenesisRetinal neovascularization was induced by expos-

ing litters of 7-day-old C57BL/6J mice with nursingdams to 75% oxygen for 5 days and then returningto room air at P12 for 5 days. Vascular nuclei abovethe internal limiting membrane (ILM) were counted,as previously described,21 with some modification. Ap-proximately 24 serial sections 4 μm thick were cutfrom each eye. Five sections, 50 μm apart, on eachside of the optic nerve were counted for neovascular-ization. Sections that included the optic nerve wereexcluded. Differences between the numbers of nucleiabove the ILM were compared. At P12, after returnto room air, mice were divided into three groups.The first group (n = 10) received intraperitoneal in-jection of 1.75 mg suramin (Sigma-Aldrich, St Louis,MO, USA) (in a solution of 0.1 ml 0.9% sodium chlo-ride). The second group (n = 19) received intraperi-toneal injection of 0.6 mg pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS)(Sigma-Aldrich, St Louis, MO, USA) (in a solution of

S. Sarman et al. 286

Dow

nloa

ded

By:

[EB

SC

OH

ost E

JS C

onte

nt D

istri

butio

n] A

t: 13

:09

26 M

arch

200

8

FIGURE 1 Expression of P2X and P2Y receptors in normal mice and in a model of ischemia-induced pathological retinal angiogenesisas assessed by immunohistochemistry. (A) P2X2 receptor expression was found in the outer plexiform layer of control mice. (B) In micetreated with oxygen, the expression was similarly found in the outer plexiform layer. In addition, a strong signal was also found in theinner plexiform layer. This induction was noted in all slides analyzed. (C) Retina without primary antibody for P2X2. (D) In control mice,weak P2Y2 receptor expression was seen in the ganglion cell and in the nerve fiber layer. (E) In mice treated with oxygen, P2Y2 expressionshowed a similar overall distribution as control mice. The signal was, however, strongly up-regulated. This increase was noted in all slidesanalyzed. (F) Retina without primary antibody for P2Y2. Black bar = 20 μm.

0.1 ml 0.9% sodium chloride). The third group (n = 14)that did not get any treatment served as control. The op-timal dose of suramin and PPADS used was estimatedfrom previous murine studies. An intraperitoneal doseof 5 mg suramin has been shown to be efficient andnon-toxic in adult mice.22 This should roughly corre-

spond to the 1.75-mg dose used in the current studyon neonatal mice. We similarly used a dose of 0.6 mgPPADS which corresponds to a dose of 140 μmol/kgused in previous studies on adult mice.23 No evidenttoxic effects of suramin or PPADS were observed onthe retina or systemically in our study.

287 P2 Receptors in Oxygen-Induced Retinopathy

Dow

nloa

ded

By:

[EB

SC

OH

ost E

JS C

onte

nt D

istri

butio

n] A

t: 13

:09

26 M

arch

200

8

FIGURE 2 Nuclei inside the ILM as evaluated in histologic sections stained with hematoxylin-eosin. Histologic sections at P17 showingneovascular nuclei inside the ILM in (A) controls, (B) suramin injected, and (C) PPADS injected mice (black arrows). Black bar = 50 μm.

Statistical AnalysisData was presented as mean ± standard error of

the mean (SEM). Statistical analysis was performedusing one-way ANOVA test after logarithmic transfor-mation of the mean nuclear value. A post hoc leastsignificance difference (LSD) test was run to establishwhich means differed. The level of significance was setat 0.05, and the analysis was performed with the SPSSsoftware (version 15.0, SPSS Inc., Chicago, IL, USA).

RESULTSExpression of P2 Receptors

in the Mouse RetinaThe expression of P2X and P2Y receptors was inves-

tigated in normal mice and in the mouse OIR modelusing immunohistochemistry. P2X2 receptor expres-sion was mainly found in the outer plexiform layer ofcontrol mice (Fig. 1A). In mice treated with oxygen,the expression was similarly found in the outer plexi-form layer. In addition, a strong signal was also found inthe inner layers, particularly the inner plexiform layer(Fig. 1B). Immunoreactivity to P2X1 and P2X3 was notseen in either control or oxygen-treated animals (datanot shown). In control mice, weak P2Y2 receptor expres-sion was seen in the ganglion cell and in the nerve fiberlayer (Fig. 1D). In mice treated with oxygen, P2Y2 ex-pression showed a similar overall distribution as controlmice. The signal was, however, strongly up-regulated(Fig. 1E).

The Effect of Treatment with P2Receptor Antagonists on Pathologic

Retinal AngiogenesisThe role of P2 receptors in pathologic retinal an-

giogenesis was investigated using the two P2 receptor

antagonists, suramin and PPADS. In untreated controlmice, the mean number of nuclei above the ILM was25.2 ± 4.8 (Fig. 2A). Systemic treatment with a singledose of either suramin (three experiments) (Fig. 2B) orPPADS (two experiments) (Fig. 2C) resulted in a dra-matic decrease in neovascularization (5.7 ± 4.2 and2.3 ± 0.5 nuclei, respectively) (Fig. 3). The effect ofsuramin on P2X2 and P2Y2 receptor expression inoxygen-treated mice was further investigated by im-munofluorescence. Intraperitoneal injection of suraminat P12 was found to down-regulate the P2X2 receptorexpression in the IPL, whereas the expression in theOPL remained unchanged compared to non-suramin-injected mice (Figs. 3B and 3C). By contrast, P2Y2receptor expression did not show any difference be-tween suramin-treated or non-treated mice (data notshown).

DISCUSSIONIn the present article, we have shown that the P2

receptors P2X2 and P2Y2 are expressed in OIR in themouse and that the pathologic retinal neovasculariza-tion was impaired by the two P2 antagonists, suraminand PPADS. We also show that suramin down-regulatesP2X2 receptors in OIR. To the best of our knowledge,the present study is the first to suggest a role for P2receptors in the development of pathologic retinal neo-vascularization.

Using immunohistochemistry, we found expres-sion of P2X2 mainly in the outer plexiform layer ofuntreated newborn mice. After oxygen treatment, wefound a marked induction of P2X2 immunoreactiv-ity in the inner layers, in particular the inner plex-iform layer. Previous studies in adult mice and ratshave shown positive immunostaining for P2X2 in theinner retinal layers, including the ganglion cell layer,the inner plexiform layer, and the inner nuclear layer,

S. Sarman et al. 288

Dow

nloa

ded

By:

[EB

SC

OH

ost E

JS C

onte

nt D

istri

butio

n] A

t: 13

:09

26 M

arch

200

8

FIGURE 3 Pathologic retinal angiogenesis induced in control (n = 14), suramin injected (n = 10), and PPADS injected (n = 19) mice.The number of nuclei above the ILM was strongly reduced after treatment with either suramin (5.7 ± 4.2 nuclei) or PPADS (2.3 ± 0.5 nuclei)as compared to untreated control mice (25.2 ± 4.8 nuclei). Pathologic retinal angiogenesis was significantly inhibited in the suramin- andPPADS-injected mice. Two asterisks indicate statistical difference between the groups (one-way ANOVA, p < 0.01). (A) Data bars representmean ± SEM of the number of neovascular nuclei on the vitreal side of the ILM. (B) Expression of P2X2 receptors in pathological retinalangiogenesis, with and without intraperitoneal injection of suramin, was assessed by immunofluorescense. P2X2 receptor expressionwas mainly found in the IPL and OPL of control mice treated only with oxygen. (C) P2X2 receptor expression in mice treated with oxygenand intraperitoneal suramin was largely confined to the OPL. Scale bar = 70 μm.

resembling the induced expression we found after oxy-gen treatment.24−26 The apparent discrepancy betweenour results and previous data regarding the P2X2 ex-pression in the outer plexiform layer is not entirelyclear. The antiserum used in this and previous stud-ies has been raised against the same peptide sequenceof the P2X2 protein, making a difference in speci-ficity less unlikely. Also, comparative studies on anti-sera from different manufacturers against this sequencehave yielded similar results.26 Based on this, the most

likely explanation for the observed P2X2 expression inthe outer plexiform layer is that it reflects the imma-ture state of the retina of the newborn mice used in thepresent study.

In agreement with previous studies demonstratingP2Y2 immunostaining mainly in the innermost reti-nal layers in adult rat and pig retina, we found ex-pression of P2Y2 receptors in the ganglion cell andnerve fiber layer of untreated and oxygen-treated new-born mice.7,27 Studies in rats using single-cell RT-PCR

289 P2 Receptors in Oxygen-Induced Retinopathy

Dow

nloa

ded

By:

[EB

SC

OH

ost E

JS C

onte

nt D

istri

butio

n] A

t: 13

:09

26 M

arch

200

8

have furthermore demonstrated that ganglion cells arelargely P2Y2 positive, whereas bipolar and Muller cellsare less frequently positive.28 Our study thus confirmsthat P2Y2 is mainly expressed in the inner retina andalso suggests that this expression is largely preservedthrough its developmental stages.

We found that P2X2 expression was induced in theinner plexiform layer and that P2Y2 expression was up-regulated in the ganglion and nerve fiber layers follow-ing oxygen treatment. This is in agreement with resultsby Cavaliere et al., showing up-regulation of P2X2 re-ceptors by hypoxia in cultures from the hippocampus.29

These ischemic conditions induced specific neuronalloss that could be prevented by the P2 receptor antag-onist suramin.30

Morigiwa et al. found that the mitotic activation in-duced by hypoxia in retinal microglia coincided withP2Y2 expression and that the mitotic activity was sup-pressed by suramin.16

We found that, in the OIR model, retinal neovas-cularization was strongly inhibited by systemic admin-istration of the P2 receptor antagonists, suramin andPPADS.

Suramin is a non-selective antagonist of native P2Xand P2Y receptors. Suramin blocks P2X2 but is onlya weak P2Y2 antagonist.4 In addition to its P2 antag-onistic effects, suramin inhibits angiogenesis by inter-fering with vascular endothelial growth factor (VEGF)and fibroblast growth factor-2 (FGF-2).30,31 PPADS, likesuramin, is a non-selective P2X and P2Y antagonist witha small preference for P2X receptors. It blocks P2X2 butis only a weak antagonist of P2Y2.4 Unlike suramin,PPADS seems to be a much more specific P2 antagonistwith minimal effects on unrelated proteins.4 We cannotrule out that the effects of suramin and PPADS on reti-nal neovascularization in the OIR model may, at leastin part, be due to inhibition of non-P2 proteins. On theother hand, the fact that we observed similar inhibitionwith two unrelated antagonists suggests that the effectsare largely mediated through P2 receptor blockade. Thisconclusion is further supported by our observation thatsuramin down-regulates P2X2 receptor expression in theIPL in OIR. The fact that expression of P2X2 receptorsin the IPL was not found in normal mice but only inOIR is interesting and suggests that P2X2 receptors inthe IPL may be directly involved in the developmentof neovascularization during OIR.

In summary, we have shown that the P2 receptorsP2X2 and P2Y2 are overexpressed in OIR in the mouse

and that the pathologic retinal neovascularization is im-paired by the two P2 antagonists, suramin and PPADS.P2 receptors may therefore be a potential new targetfor treatment of neovascular retinopathies, includingretinopathy of prematurity and diabetic retinopathy.

ACKNOWLEDGMENTSThe authors thank Monica Aronsson, Berit

Spangberg, Margareta Oskarsson, German Ruffolo,and Anne Winter Wernersson for excellent technicalsupport. This study was supported by grants fromKarolinska Institutet, the Crown Princess MargaretasFoundation (KMA), Sigvard and Marianne BernadottesResearch Foundation for Children’s Eye Care, Stif-telsen Synframjandets Forskningsfond, Stockholm,Sweden, and by a grant from Austral University.Financial support was also provided through theregional agreement on medical training and clinicalresearch (ALF) between Stockholm County Counciland Karolinska Institutet.

REFERENCES[1] Lazarowski E, Boucher R, Harden T. Mechanisms of release of nu-

cleotides and integration of their action as P2X- and P2Y-receptoractivating molecules. Mol Pharmacol. 2003;64:785–795.

[2] P2 Receptors. Sigma-Aldrich.com/ehandbook. Rev 2002;8:64–67.

[3] P2 Receptors: P2Y G-Protein Family. Sigma-Aldrich.com/ehandbook.Rev 2002;8:134–137.

[4] Lambrecht G, Braun K, Damer S, Ganso M, Hildebrandt C, UllmanH, Kassack M, Nickel P. Structure-activity relationships of suramin, apyridoxal-5′-phosphate derivative as P2 receptor antagonists. CurrPharmaceut Des. 2002;8:2371–2399.

[5] Neary JT, Rathbone MP, Cattabeni F, Abbracchio MP, Burnstock G.Trophic actions of extracellular nucleotides and nucleosides on glialand neuronal cells. Trends Neurosci. 1996;19:13–18.

[6] Sugioka M, Fukuda Y, Yamashita M. Ca2+ responses to ATPvia purinoceptors in the early embryonic chick retina. J Physiol.1996;493:855–863.

[7] Ianiev I, Uckermann O, Pannicke T, Wurm A, Tenckhoff S, Pietsch U,Reichenbach A, Wiedemann P, Bringmann A, Uhlmann S. Glial cellreactivity in a porcine model of retinal detachment. Invest Ophthal-mol Vis Sci. 2006;47:2161–2171.

[8] Francke M, Weick M, Pannicke T, Uckermann O, Grosche J, GoczalikI, Milenkovic I, Uhlmann S, Faude F, Wiedemann P, Reichenbach A,Bringmann A. Up-regulation of extracellular ATP-induced Muller cellresponses in a dispase model of proliferative vitreoretinopathy. InvestOphthalmol Vis Sci. 2002;43:870–881.

[9] Sugiyama T, Kabayashi M, Kawamura H, Li Q, Puro D. Enhancementof P2X7-induced pore formation and apoptosis: An early effect ofdiabetes on the retinal microvasculature. Invest Ophthalmol Vis Sci.2004;45:1026–1032.

[10] Sugiyama T, Oku H, Komori A, Ikeda T. Effect of P2X7 receptor ac-tivation on the retinal blood velocity of diabetic rabbits. Arch Oph-thalmol. 2006;124:1143–1149.

[11] Sugiyama T, Kawamura H, Yamanishi S, Kobayashi M, Katsumura K,Puro D. Regulation of P2X7-induced pore formation and cell death

S. Sarman et al. 290

Dow

nloa

ded

By:

[EB

SC

OH

ost E

JS C

onte

nt D

istri

butio

n] A

t: 13

:09

26 M

arch

200

8

in pericyte-containing retinal microvessels. Am J Physiol Cell Physiol.2005;288:C568–C576.

[12] Francke H, Klimke K, Brinckmann U, Grosche J, Francke M, SperlaghB, Reichenbach A, Liebert UG, Illes P. P2X(7) receptor-mRNA andprotein in the mouse retina; changes during retinal degeneration inBALBCrds mice. Neurochem Int. 2005;47:235–242.

[13] Bucolo C, Marrazzo A, Ronsisvalle S, Ronsisvalle G, Cuzzocrea S,Mazzon E, Caputi A, Drago F. A novel adamantane derivative atten-uates retinal ischemic-reperfusion damage in the rat retina throughσ1 receptors. Eur J Pharmacol. 2006;536:200–203.

[14] Osborne N, Schwarz M, Pergande G. Protection of rabbit retinafrom ischemic injury by flupirtine. Invest Ophthalmol. Vis Sci.1996;37:274–280.

[15] Siesjo BK. Pathophysiology and treatment of focal cerebral ischemia.Part I: Pathophysiology. J Neurosurg. 1992;77:169–184.

[16] Morigiwa K, Quan M, Murakami M, Yamashita M, FukudaY. P2 purinoceptor expression and functional changes ofhypoxia-activated cultured rat retinal microglia. Neurosci Lett.2002;282:153–156.

[17] Fruttiger M. Development of the mouse retinal vasculature:Angiogenesis versus vasculogenesis. Invest Ophthalmol Vis Sci.2002;43:522–527.

[18] Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J.Vascular-specific growth factors and blood vessel formation. Nature.2000;407:249–257.

[19] Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases.Nature. 2000;407:249–257.

[20] Carmeliet P. Angiogenesis in health and disease. Nat Med.2003;9:653–660.

[21] Smith L, Wesolowski E, McLellan A, Kostyk S, D’Amato R, Sullivan R,D’Amore P. Oxygen-induced retinopathy in the mouse. Invest Oph-thalmol Vis Sci. 1994;35:101–111.

[22] Eichhorst S, Krueger A, Muerkoster S, Fas S, Golks A, Gruetzner U,Schubert L, Opelz C, Bilzer M, Gerbes A, Krammer P. Suramin inhibits

death receptor-induced apoptosis in vitro and fulminant apoptoticliver damage in mice. Nat Med. 2004;10:602–609.

[23] Honore P, Mikusa J, Bianchi B, McDonald H, Cartmell J, Faltynek C,Jarvis M. TNP-ATP, a potent P2X3 receptor antagonist, blocks aceticacid-induced abdominal constriction in mice: Comparison with ref-erence analgesics. Pain. 2002;96:99–105.

[24] Greenwood D, Yao W, Housley G. Expression of the P2X2 receptorsubunit of the ATP-gated ion channel in the retina. Neuroreport.1997;8:1083–1088.

[25] Kaneda M, Ishii K, Morishima Y, Akagi T, Yamazaki Y, NakanishiS, Hashikawa T. OFF-cholinergic-pathway-selective localizationof P2X2 purinoceptors in the mouse retina. J Comp Neurol.2004;476:103–111.

[26] Puthussery T, Fletcher E. P2X2 receptors on ganglion and amacrinecells in cone pathways of the rat retina. J Comp Neurol.2006;496:595–609.

[27] Fries J, Wheeler-Schilling T, Guenther E, Kohler K. Expression ofP2Y1, P2Y2, P2Y4 and P2Y6 receptor subtypes in the rat retina.Invest Ophthalmol Vis Sci. 2004;45:3410–3417.

[28] Fries J, Wheeler-Schilling T, Kohler K, Guenther E. Distribution ofmetabotropic P2Y receptors in the rat retina: A single-cell RT-PCRstudy. Mol Brain Res. 2004;130:1–6.

[29] Cavaliere F, Florenzano F, Amadio S, Fusco F, Viscomi M, D’AmbrosiN, Vacca F, Sancesario G, Bernardi G, Molinari M, VolonteC. Up-regulation of P2X2, P2X4 receptor and ischemic celldeath: Prevention by P2 antagonists. Neuroscience. 2003;120:85–98.

[30] Waltenberger J, Mayr U, Frank H, Hombach V. Suramin is a potentinhibitor of vascular endothelial growth factor. A contribution tothe molecular basis of its antiangiogenic action. J Mol Cell Cardiol.1996;28:1523–1529.

[31] Kathir K, Kumar T, Yu C. Understanding the mechanism ofthe antimitogenic activity of suramin. Biochemistry. 2006;45:899–906.

291 P2 Receptors in Oxygen-Induced Retinopathy