Dramatic Inhibition of Retinal and Choroidal Neovascularization by Oral Administration of a Kinase Inhibitor
1999; Elsevier BV; Volume: 154; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65430-2
ISSN1525-2191
AutoresMan Seong Seo, Nohoon Kwak, Hiroaki Ozaki, Haruhiko Yamada, Naoyuki Okamoto, Eri Yamada, Doriano Fabbro, Francesco Hofmann, Jeanette M. Wood, Peter A. Campochiaro,
Tópico(s)Retinal Development and Disorders
ResumoThe most common cause of new blindness in young patients is retinal neovascularization, and in the elderly is choroidal neovascularization. Therefore, there has been a great deal of attention focused on the development of new treatments for these disease processes. Previous studies have demonstrated partial inhibition of retinal neovascularization in animal models using antagonists of vascular endothelial growth factor or other signaling molecules implicated in the angiogenesis cascade. These studies have indicated potential for drug treatment, but have left many questions unanswered. Is it possible to completely inhibit retinal neovascularization using drug treatment with a mode of administration that is feasible to use in patients? Do agents that inhibit retinal neovascularization have any effect on choroidal neovascularization? In this study, we demonstrate complete inhibition of retinal neovascularization in mice with oxygen-induced ischemic retinopathy by oral administration of a partially selective kinase inhibitor that blocks several members of the protein kinase C family, along with vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases. The drug also blocks normal vascularization of the retina during development but has no identifiable adverse effects on mature retinal vessels. In addition, the kinase inhibitor causes dramatic inhibition of choroidal neovascularization in a laser-induced murine model. These data provide proof of concept that pharmacological treatment is a viable approach for therapy of both retinal and choroidal neovascularization. The most common cause of new blindness in young patients is retinal neovascularization, and in the elderly is choroidal neovascularization. Therefore, there has been a great deal of attention focused on the development of new treatments for these disease processes. Previous studies have demonstrated partial inhibition of retinal neovascularization in animal models using antagonists of vascular endothelial growth factor or other signaling molecules implicated in the angiogenesis cascade. These studies have indicated potential for drug treatment, but have left many questions unanswered. Is it possible to completely inhibit retinal neovascularization using drug treatment with a mode of administration that is feasible to use in patients? Do agents that inhibit retinal neovascularization have any effect on choroidal neovascularization? In this study, we demonstrate complete inhibition of retinal neovascularization in mice with oxygen-induced ischemic retinopathy by oral administration of a partially selective kinase inhibitor that blocks several members of the protein kinase C family, along with vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases. The drug also blocks normal vascularization of the retina during development but has no identifiable adverse effects on mature retinal vessels. In addition, the kinase inhibitor causes dramatic inhibition of choroidal neovascularization in a laser-induced murine model. These data provide proof of concept that pharmacological treatment is a viable approach for therapy of both retinal and choroidal neovascularization. The retina receives its blood supply from two vascular beds: retinal vessels, which supply the inner two-thirds of the retina, and choroidal vessels, which supply the outer one-third. Damage to retinal blood vessels resulting in closure of retinal capillaries and retinal ischemia occurs in several disease processes, including diabetic retinopathy, retinopathy of prematurity, branch retinal vein occlusion, and central retinal vein occlusion; they are collectively referred to as ischemic retinopathies. Retinal ischemia results in release of one or more angiogenic factors that stimulate neovascularization. The new vessels break through the internal limiting membrane that lines the inner surface of the retina and grow along the outer surface of the vitreous. They recruit many other cells and produce sheets of vessels, cells, and extracellular matrix that exert traction on the retina, often leading to retinal detachment and severe loss of vision. Panretinal laser photocoagulation increases oxygenation in the retina and can result in involution of neovascularization.1Pournaras CJ Tsacopoulos M Strommer K Gilodi N Leuenberger PM Scatter photocoagulation restores tissue hypoxia in experimental vasoproliferative microangiopathy in miniature pigs.Ophthalmology. 1990; 97: 1329-1333Abstract Full Text PDF PubMed Scopus (68) Google Scholar However, despite the effectiveness of laser photocoagulation,2The Diabetic Retinopathy Research Study Group Photocoagulation treatment of proliferative diabetic retinopathy: clinical application of Diabetic Retinopathy Study (DRS) findings, DRS Report Number 8.Ophthalmology. 1981; 88: 583-600Abstract Full Text PDF PubMed Scopus (987) Google Scholar diabetic retinopathy remains the most common cause of severe vision loss in patients less than 60 years of age in developed countries, and therefore additional treatments are needed. Choroidal neovascularization occurs in several diseases in which there are abnormalities of Bruch's membrane. The most prevalent disease of this type is age-related macular degeneration, the most common cause of severe vision loss in patients over the age of 60 in developed countries.3Macular Photocoagulation Study Group Argon laser photocoagulation for neovascular maculopathy: five year results from randomized clinical trials.Arch Ophthalmol. 1991; 109: 1109-1114Crossref PubMed Scopus (644) Google Scholar Neovascularization originating from choroidal vessels grows through Bruch's membrane into the sub-retinal pigmented epithelial space and sometimes into the subretinal space. The blood vessels leak fluid, which collects beneath the retina causing reversible visual loss, and they bleed and cause scarring that results in permanent loss of central vision. Current treatments are designed to destroy or remove the abnormal blood vessels and do not address the underlying stimuli responsible for neovascularization; therefore, recurrent neovascularization and permanent visual loss occur in the majority of patients who initially have successful treatment.3Macular Photocoagulation Study Group Argon laser photocoagulation for neovascular maculopathy: five year results from randomized clinical trials.Arch Ophthalmol. 1991; 109: 1109-1114Crossref PubMed Scopus (644) Google Scholar Drug treatment that blocks the stimuli for choroidal neovascularization would be a major advance, but its development is hindered by our poor understanding of pathogenesis. More is known about the cascade of events leading to retinal neovascularization than that for choroidal neovascularization, because some of the molecular signals involved in the development of retinal neovascularization have been defined. For instance, several lines of evidence suggest that vascular endothelial growth factor (VEGF) plays an important role in retinal vascularization during development and in pathological neovascularization in ischemic retinopathies. The expression of VEGF is increased by hypoxia,4Forsythe JA Jiang B-H Iyer NV Agani F Leung SW Koos RD Semenza G Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.Mol Cell Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3135) Google Scholar, 5Levy AP Levy NS Wegner S Goldberg MA Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia.J Biol Chem. 1995; 270: 13333-13340Crossref PubMed Scopus (875) Google Scholar which is a prominent feature of both of these processes. Stimulated by VEGF released by the avascular, hypoxic peripheral retina, blood vessels begin to develop at the optic nerve and extend to the periphery of the retina.6Stone J Itin A Alon T Pe'er J Gnessin H Chan-Ling T Keshet E Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia.J Neurosci. 1995; 15: 4738-4747Crossref PubMed Google Scholar Likewise, VEGF participates in pathological retinal neovascularization, because its levels are increased in the retina and vitreous of patients7Adamis AP Miller JW Bernal M-T D'Amico DJ Folkman J Yeo T-K Yeo K-T Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy.Am J Ophthalmol. 1994; 118: 445-450Abstract Full Text PDF PubMed Scopus (1194) Google Scholar, 8Aiello LP Avery RL Arrigg PG Keyt BA Jampel HD Shah ST Pasquale LR Thieme H Iwamoto MA Park JE Nguyen MS Aiello LM Ferrara N King GL Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders.N Engl J Med. 1994; 331: 1480-1487Crossref PubMed Scopus (3343) Google Scholar, 9Malecaze F Clamens S Simorre-Pinatel V Mathis A Chollet P Favard C Bayard F Plouet J Detection of vascular endothelial growth factor messenger RNA and vascular endothelial growth factor-like activity in proliferative diabetic retinopathy.Arch Ophthalmol. 1994; 112: 1476-1482Crossref PubMed Scopus (339) Google Scholar, 10Pe'er J Shweiki D Itin A Hemo I Gnessin H Keshet E Hypoxia-induced expression of vascular endothelial growth factor by retinal cells is a common factor in neovascularizing ocular diseases.Lab Invest. 1995; 72: 638-645PubMed Google Scholar or laboratory animals11Miller JW Adamis AP Shima DT D'Amore PA Moulton RS O'Reilly MS Folkman J Dvorak HF Brown LF Berse B Yeo T-K Yeo K-T Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model.Am J Pathol. 1994; 145: 574-584PubMed Google Scholar, 12Pierce EA Avery RL Foley ED Aiello LP Smith LEH Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization.Proc Natl Acad Sci USA. 1995; 92: 905-909Crossref PubMed Scopus (937) Google Scholar with ischemic retinopathies, and increased expression of VEGF in retinal photoreceptors of transgenic mice stimulates neovascularization within the retina.13Okamoto N Tobe T Hackett SF Ozaki H Vinores MA LaRochelle W Zack DJ Campochiaro PA Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization.Am J Pathol. 1997; 151: 281-291PubMed Google Scholar The implication of VEGF in retinal neovascularization led to studies investigating VEGF antagonists in models of ischemic retinopathy. Soluble VEGF receptor/IgG fusion proteins or VEGF antisense oligonucleotides each inhibited retinal neovascularization by ∼50% in the murine model of oxygen-induced ischemic retinopathy.14Aiello LP Pierce EA Foley ED Takagi H Chen H Riddle L Ferrara N King GL Smith LEH Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.Proc Natl Acad Sci USA. 1995; 92: 10457-10461Crossref PubMed Scopus (1152) Google Scholar, 15Robinson GS Pierce EA Rook SL Foley E Webb R Smith LES Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.Proc Natl Acad Sci USA. 1996; 93: 4851-4856Crossref PubMed Scopus (349) Google Scholar Antibodies to VEGF partially inhibited iris neovascularization in a monkey model of ischemic retinopathy.16Adamis AP Shima DT Tolentino MJ Gragoudas ES Ferrara N Folkman J D'Amore PA Miller JW Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization.Arch Ophthalmol. 1996; 114: 66-71Crossref PubMed Scopus (571) Google Scholar Although VEGF plays a central role, it is not the only stimulator involved, which might explain why VEGF antagonists are only partially effective. Growth hormone acting through insulin-like growth factor (IGF)-I also participates in retinal neovascularization, and decreased IGF-I in genetically engineered mice or antagonism of IGF-I by somatostatin analogs results in approximately a 30% decrease in retinal neovascularization in mice with ischemic retinopathy.17Smith LEH Kopchick JJ Chen W Knapp J Kinose F Daley D Foley E Smith RG Schaeffer JM Essential role of growth hormone in ischemia-induced retinal neovascularization.Science. 1997; 276: 1706-1709Crossref PubMed Scopus (373) Google Scholar Intracellular signaling induced by VEGF is complex, but it has been suggested that protein kinase C (PKC), particularly the PKCβII isoform, plays a prominent role.18Inoguchi T Battan R Handler R Sportsman JR Heath W King GL Preferential elevation of protein kinase C isoform β II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation.Proc Natl Acad Sci USA. 1992; 89: 11059-11063Crossref PubMed Scopus (696) Google Scholar, 19Aiello LP Bursell S-E Clermont A Duh E Ishii H Takagi C Mori F Ciulla TA Ways K Jirousek M Smith LEH King GL Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective β-isoform-selective inhibitor.Diabetes. 1997; 46: 1473-1480Crossref PubMed Google Scholar A specific antagonist of PKCβ isoforms partially inhibits retinal neovascularization after laser-induced branch vein occlusion.20Danis RP Bingaman DP Jirousek M Yang Y Inhibition of intraocular neovascularization caused by retinal ischemia in pigs by PKCβ inhibition with LY 333531.Invest Ophthalmol Vis Sci. 1998; 39: 171-179PubMed Google Scholar Integrins αvβ3 and αvβ5 are induced on endothelial cells, including those in the retina, participating in neovascularization.21Brooks P Clark R Cheresh D Requirement of vascular integrin α-v β-3 for angiogenesis.Science. 1994; 264: 569-571Crossref PubMed Scopus (2704) Google Scholar, 22Luna J Tobe T Mousa SA Reilly TM Campochiaro PA Antagonists of integrin α-v β-3 inhibit retinal neovascularization in a murine model.Lab Invest. 1996; 75: 563-573PubMed Google Scholar Two independent studies using different peptides that antagonize binding to αvβ3 or both αvβ3 and αvβ5 each demonstrated up to 50% inhibition of retinal neovascularization in the murine model of oxygen-induced ischemic retinopathy.22Luna J Tobe T Mousa SA Reilly TM Campochiaro PA Antagonists of integrin α-v β-3 inhibit retinal neovascularization in a murine model.Lab Invest. 1996; 75: 563-573PubMed Google Scholar, 23Hammes H Brownlee M Jonczyk A Sutter A Preissner K Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization.Nature Med. 1996; 2: 529-533Crossref PubMed Scopus (312) Google Scholar Thus, several different types of agents that work by different mechanisms can cause partial inhibition of retinal neovascularization. This suggests that drug treatment of retinal neovascularization in patients may be feasible, but there are several questions related to this issue that remain. For instance, why is it that 50% inhibition has been the maximal achievable limit with several types of agents given by different routes of administration, including intraocular injections? Is there so much redundancy built into the retinal neovascularization cascade that this is the most that can be attained, and if so, is it sufficient to provide clinical benefit? Would an agent or combination of agents that act on multiple targets in the cascade be more effective? Do any of the molecular signals implicated in retinal neovascularization also play a role in choroidal neovascularization, and are there agents that inhibit both? PKC consists of a family of at least 10 related serine/threonine kinases.24Nishizuka Y The molecular heterogeneity of protein kinase C and its implications for cellular regulation.Nature. 1988; 334: 661-665Crossref PubMed Scopus (3521) Google Scholar Staurosporine is an alkaloid produced by bacteria that is a potent nonspecific inhibitor of PKC25Tamaoki T Hisayo N Takahashi I Kato Y Morimoto M Tomita F Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase.Biochem Biophys Res Commun. 1986; 135: 397-402Crossref PubMed Scopus (2205) Google Scholar that also inhibits other serine/threonine kinases, such as protein kinase A (PKA), and tyrosine kinases, such as epidermal growth factor receptor (EGFR).26Meyer T Regenass U Fabbro D Alteri E Rosel J Muller M Caravatti G Matter A A derivative of staurosporin (CGP 41 251) shows selectivity for protein kinase C inhibition and in vitro anti-proliferative as well as in vivo anti-tumor activity.Int J Cancer. 1989; 43: 851-856Crossref PubMed Scopus (432) Google Scholar CGP 41251 is a derivative of staurosporine with the chemical name N-benzoyl-staurosporine that was developed as a PKC inhibitor for treatment of cancer.26Meyer T Regenass U Fabbro D Alteri E Rosel J Muller M Caravatti G Matter A A derivative of staurosporin (CGP 41 251) shows selectivity for protein kinase C inhibition and in vitro anti-proliferative as well as in vivo anti-tumor activity.Int J Cancer. 1989; 43: 851-856Crossref PubMed Scopus (432) Google Scholar It is a less potent inhibitor of PKC than staurosporine but is more specific because it is a weak inhibitor of PKA and EGFR, and it has been used in several studies to assess the role of PKC in cellular functions.27Miggli V Keller H On the role of protein kinases in regulating neutrophil actin association with the cytoskeleton.J Biol Chem. 1991; 266: 7927-7932PubMed Google Scholar, 28Chung DL Brandt-Rauf PW Weinstein IB Nishimura S Yamaizumi Z Murphy RB Pincus MR Evidence that the ras oncogene-encoded p21 protein induces oocyte maturation via activation of protein kinase C.Proc Natl Acad Sci USA. 1992; 89: 1993-1996Crossref PubMed Scopus (39) Google Scholar, 29Marte BM Meyer T Stabel S Standke GJR Jaken S Fabbro D Hynes NE Protein kinase C and mammary cell differentiation: involvement of protein kinase C α in the induction of β-casein expression.Cell Growth Differ. 1994; 5: 239-247PubMed Google Scholar, 30Oh LYS Goodyer CG Olivier A Yong VW The promoting effects of bFGF and astrocyte extracellular matrix on process outgrowth by adult human oligodendrocytes are mediated by protein kinase C.Brain Res. 1997; 757: 236-244Crossref PubMed Scopus (14) Google Scholar Recently, one of the authors (J.M. Wood) determined that CGP 41251 is also a relatively potent inhibitor of VEGF and platelet-derived growth factor (PDGF) receptor tyrosine kinases (unpublished data). It was also shown to inhibit VEGF-induced angiogenesis in a mouse subcutaneous growth factor implant model. As antagonism of VEGF and inhibition of PKC, each have been demonstrated to partially inhibit retinal neovascularization14Aiello LP Pierce EA Foley ED Takagi H Chen H Riddle L Ferrara N King GL Smith LEH Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.Proc Natl Acad Sci USA. 1995; 92: 10457-10461Crossref PubMed Scopus (1152) Google Scholar, 15Robinson GS Pierce EA Rook SL Foley E Webb R Smith LES Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.Proc Natl Acad Sci USA. 1996; 93: 4851-4856Crossref PubMed Scopus (349) Google Scholar, 20Danis RP Bingaman DP Jirousek M Yang Y Inhibition of intraocular neovascularization caused by retinal ischemia in pigs by PKCβ inhibition with LY 333531.Invest Ophthalmol Vis Sci. 1998; 39: 171-179PubMed Google Scholar and CGP 41251 has both activities, we investigated the effect of CGP 41251 in animal models of retinal and choroidal neovascularization. The effect of CGP 41251 on the enzymatic activity of several members of the PKC family was measured using purified enzymes and artificial substrates as previously described.29Marte BM Meyer T Stabel S Standke GJR Jaken S Fabbro D Hynes NE Protein kinase C and mammary cell differentiation: involvement of protein kinase C α in the induction of β-casein expression.Cell Growth Differ. 1994; 5: 239-247PubMed Google Scholar The effect of CGP 41251 on phosphorylation of VEGFRs and other tyrosine kinase receptors was measured using purified recombinant glutathione S-transferase (GST)-fused kinase domains in the presence of substrate and labeled ATP.31Meggio F Donella-Deana A Ruzzene M Brunati AM Cesaro L Guerra B Meyer T Mett H Fabbro D Furet P Dobrowolska G Pinna LA Different susceptibility of protein kinases to staurosporine inhibition: kinetic studies and molecular bases for the resistance of protein kinase CK2.Eur J Biochem. 1995; 234: 317-322Crossref PubMed Scopus (244) Google Scholar The kinase domain-fusion proteins were expressed in baculovirus, purified over glutathione-Sepharose, and diluted in 10 mmol/L Tris/HCl (pH 7.2) based on their specific activity to obtain an activity of 4000 to 6000 cpm above background (<400 cpm). [33P]ATP (Amersham, Arlington Heights, IL) was used as the phosphate donor, and the polyGluTyr(4:1) peptide (P-275, Sigma Chemical Co., St. Louis, MO) was used as the acceptor. CGP 41251 was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mmol/L and then diluted as required so that the final DMSO concentration was 1%. The assay mixture, which was optimized for each kinase (20 mmol/L Tris/HCl (pH 7.5), 1 to 10 mmol/L MnCl2, 1 to 10 mmol/L MgCl2, 1 to 8 μmol/L ATP, 0.2 μCi of [33P]ATP, 3 to 8 μg/ml polyGluTyr(4:1)) was incubated with the respective GST-fused kinase with or without CGP 41251 for 10 minutes at room temperature in a total volume of 30 μl. The reaction was stopped by adding 10 μl of 250 mmol/L EDTA. Using a 96-well filter system (GIBCO BRL, Gaithersburg, MD), 20 μl of the reaction mixture was transferred onto an Immobilon-PVDF membrane (IPVH 000 10, Millipore, Bedford, MA). Membranes were washed extensively with 0.5% H3PO4 and soaked in ethanol. After drying, Microscint cocktail (TM-0 6013611, Packard, Meriden, CT) was added, and scintillation counting was performed (Hewlett Packard Top Count). IC50 values were calculated by linear regression analysis of the percentage inhibition by CGP 41251 over a range of different concentrations Ischemic retinopathy was produced in C57BL/6J mice by a method described by Smith et al.32Smith LEH Wesolowski E McLellan A Kostyk SK D'Amato R Sullivan R D'Amore PA Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar Seven-day-old (P7) mice and their mothers were placed in an airtight incubator and exposed to an atmosphere of 75 ± 3% oxygen for 5 days. Incubator temperature was maintained at 23 ± 2°C, and oxygen was measured every 8 hours with an oxygen analyzer. After 5 days, the mice were removed from the incubator and placed in room air, and drug treatment was begun. Drug was dissolved in DMSO and diluted to final concentrations with water; the maximal concentration of DMSO was 1%. Vehicle (1% DMSO) or vehicle containing various concentrations of drug (volume = 10 μl per gram body weight) was placed in the stomach by gavage once a day. At P17, after 5 days of treatment, mice were sacrificed, and eyes were rapidly removed and frozen in optimal cutting temperature embedding compound (OCT; Miles Diagnostics, Elkhart, IN) or fixed in 10% phosphate-buffered formalin and embedded in paraffin. Adult C57BL/6J mice were also treated by gavage with drug or vehicle, and after 5 days, they were sacrificed and their eyes were processed for frozen or paraffin sections. Frozen sections (10 μm) of eyes from drug-treated and control mice were histochemically stained with biotinylated griffonia simplicifolia lectin B4 (GSA, Vector Laboratories, Burlingame, CA), which selectively binds to endothelial cells. Slides were incubated in methanol/H2O2 for 10 minutes at 4°C, washed with 0.05 mol/L Tris-buffered saline, pH 7.6 (TBS), and incubated for 30 minutes in 10% normal porcine serum. Slides were incubated for 2 hours at room temperature with biotinylated GSA, and after rinsing with 0.05 mol/L TBS, they were incubated with avidin coupled to peroxidase (Vector Laboratories) for 45 minutes at room temperature. After being washed for 10 minutes with 0.05 mol/L TBS, slides were incubated with diaminobenzidine to give a brown reaction product. Some slides were counterstained with hematoxylin, and all were mounted with Cytoseal. To perform quantitative assessments, 10-μm serial sections were cut through one-half of each eye, and sections roughly 50 to 60 μm apart were stained with GSA, providing 13 sections per eye for analysis. GSA-stained sections were examined with an Axioskop microscope (Zeiss, Thornwood, NY), and images were digitized using a 3 CCD color video camera (IK-TU40A, Toshiba, Tokyo, Japan) and a frame grabber. Image-Pro Plus software (Media Cybernetics, Silver Springs, MD) was used to delineate GSA-stained cells on the surface of the retina, and their area was measured. The mean of the 13 measurements from each eye was used as a single experimental value. Litters of newborn C57BL/6J mice were divided into treatment and control groups that received daily subcutaneous injections of 100 mg/kg drug or vehicle, respectively. At P7 or P10, mice were anesthetized and perfused with 1 ml of phosphate-buffered saline containing 50 mg/ml fluorescein-labeled dextran (2 × 106 average molecular weight; Sigma) as previously described.33Tobe T Okamoto N Vinores MA Derevjanik NL Vinores SA Zack DJ Campochiaro PA Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors.Invest Ophthalmol Vis Sci. 1998; 39: 180-188PubMed Google Scholar The eyes were removed and fixed for 1 hour in 10% phosphate-buffered formalin. The cornea and lens were removed and the entire retina was carefully dissected from the eyecup. Radial cuts were made from the edge of the retina to the equator in all four quadrants, and the retina was flat mounted in Aquamount with photoreceptors facing upward. Flat mounts were examined by fluorescence microscopy, and images were digitized using a 3 CCD color video camera and a frame grabber. Image-Pro Plus was used to measure the distance from the center of the optic nerve to the leading front of developing retinal vessels in each quadrant, and the mean was used as a single experimental value. Choroidal neovascularization was generated by modification of a previously described technique.34Tobe T Ortega S Luna L Ozaki H Okamoto N Derevjanik NL Vinores SA Basilico C Campochiaro PA Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model.Am J Pathol. 1998; 153: 1641-1646Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar Briefly, 4- to 5-week-old male C57BL/6J mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight), and the pupils were dilated with 1% tropicamide. Three burns of krypton laser photocoagulation (100-μm spot size, 0.1-second duration, 150 mW) were delivered to each retina using the slit lamp delivery system of a Coherent model 920 photocoagulator and a hand-held cover slide as a contact lens. Burns were performed in the 9, 12, and 3 o'clock positions of the posterior pole of the retina. Production of a bubble at the time of laser, which indicates rupture of Bruch's membrane, is an important factor in obtaining choroidal neovascularization,34Tobe T Ortega S Luna L Ozaki H Okamoto N Derevjanik NL Vinores SA Basilico C Campochiaro PA Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model.Am J Pathol. 1998; 153: 1641-1646Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar so only mice in which a bubble was produced for all three burns were included in the study. Ten mice were randomly assigned to treatment with vehicle alone, and 10 mice received vehicle containing 400 mg/kg/day of CGP 41251 orally by gavage. After 14 days, the mice were killed with an overdose of pentobarbital sodium, and their eyes were rapidly removed and frozen in OCT. Frozen serial sections (10 μm) were cut through the entire extent of each burn and histochemically stained with biotinylated GSA as described above. Histomark Red (Kirkegaard and Perry, Gaithersburg, MD) was used as chromogen to give a red reaction product that is distinguishable from melanin. Some slides were counterstained with Contrast Blue (Kirkegaard and Perry). To perform quantitative assessments, GSA-stained sections were examined with an Axioskop microscope, and images were digitized using a 3 CCD color video camera and a frame grabber. Image-Pro Plus software was used to delineate and measure the area of GSA-stained blood vessels in the subretinal space. For each lesion, area measurements were made for all sections on which some of the lesion appeared and added together to give the integrated area measurement. Only lesions in which good sections were obtained through the entire lesion, so that a valid area measurement could be made on each, were included in the analysis. There appeared to be little variability among lesions in individual mice, and all excluded lesions were qualitatively similar in size to included lesions and were excluded solely due to inability to obtain an accurate measurement because of poor quality of some sections. Values were averaged to give one experimental value per mouse. A two-sample t-test for unequal variances was performed to compare the log mean integrated area between treated and control mice. Table 1 shows the kinase inhibitory profile of CGP 41251. The IC50 for several subtypes of PKC as well as the KDR tyrosine kinase of human VEGF receptor-2 and the tyrosine kinase of human PDGF receptor-β are in the same range (20 to 100 nmol/L). At approximately 10-fold higher concentration
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