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ABSTRACT: Equine infectious anemia virus (EIAV) is a nonprimate lentivirus that does not cause human disease. Subretinal injection into mice of a recombinant EIAV lentiviral vector in which lacZ is driven by a CMV promoter (EIAV CMV LacZ) resulted in rapid and strong expression of LacZ in retinal pigmented epithelial (RPE) cells and some other cells including ganglion cells, resulting in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside within the optic nerve. Substitution of the RPE-specific promoter from the vitelliform macular dystrophy (VMD2) gene for the CMV promoter resulted in prolonged (at least 1 year) expression of LacZ that was restricted to RPE cells, albeit reduced 6- to 10-fold compared with the CMV promoter. Similarly, the amount of FLAG-tagged endostatin detected in eyes injected with the EIAV VMD2 Endo(FLAG) vector was similar to that seen in eyes injected with a vector that expressed both endostatin and angiostatin [EIAV VMD2 Endo(FLAG)/Angio]; expression was approximately 6-fold lower than with identical vectors in which the CMV promoter drove expression. Compared with murine eyes treated with a control EIAV vector, subretinal injection of EIAV vectors expressing murine endostatin alone or in combination with angiostatin driven by either the CMV or VMD2 promoter caused significant suppression of choroidal neovascularization (NV) at laser-induced rupture sites in Bruch's membrane. These data support proceeding toward clinical studies with EIAV-based gene therapy for choroidal NV, using the VMD2 promoter to selectively drive expression of a combination of endostatin and angiostatin in RPE cells.
Human gene therapy 01/2009; 20(1):31-9. · 4.20 Impact Factor
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ABSTRACT: Equine infectious anemia virus (EIAV) is a non-primate lentivirus that does not cause human disease. Subretinal injection in mice of a recombinant EIAV lentiviral vector in which LacZ is driven by a CMV promoter (EIAV CMV LacZ) resulted in rapid and strong expression of LacZ in retinal pigmented epithelial (RPE) cells and some other cells including ganglion cells resulting in X-gal within the optic nerve. Substitution of the RPE-specific VMD2 promoter for the CMV promoter resulted in prolonged (at least 1 year) expression of LacZ that was restricted to RPE cells albeit reduced 6-10 fold compared to the CMV promoter. Similarly, the amount of FLAG-tagged endostatin detected in eyes injected with the EIAV VMD2 Endo(FLAG) vector was similar to that seen in eyes injected with a vector that expressed both endostatin and angiostatin (EIAV VMD2 Endo(FLAG)/Angio, and was approximately 6-fold lower than identical vectors in which the CMV promoter drove expression. Compared to murine eyes treated with a control EIAV vector, subretinal injection of EIAV vectors expressing murine endostatin alone or in combination with angiostatin driven by either the CMV or VMD2 promoters caused significant suppression of choroidal neovascularization (NV) at laser-induced rupture sites in Bruchs membrane. These data support proceeding toward clinical studies with an EIAV-based gene therapy for choroidal NV using the VMD2 promoter to selectively drive expression of a combination of endostatin and angiostatin in RPE cells.
Human gene therapy 11/2008; · 4.20 Impact Factor
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John Doukas,
Sankaranarayana Mahesh,
Naoyasu Umeda,
Shu Kachi, Hideo Akiyama,
Katsutoshi Yokoi,
Jon Cao,
Zoe Chen,
Luis Dellamary,
Betty Tam,
Adrienne Racanelli-Layton,
John Hood,
Michael Martin,
Glenn Noronha,
Richard Soll,
Peter A Campochiaro
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ABSTRACT: Age-related macular degeneration, diabetic retinopathy, and retinal vein occlusions are complicated by neovascularization and macular edema. Multi-targeted kinase inhibitors that inhibit select growth factor receptor tyrosine kinases and/or components of their down-stream signaling cascades (such as Src kinases) are rationale treatment strategies for these disease processes. We describe the discovery and characterization of two such agents. TG100572, which inhibits Src kinases and selected receptor tyrosine kinases, induced apoptosis of proliferating endothelial cells in vitro. Systemic delivery of TG100572 in a murine model of laser-induced choroidal neovascularization (CNV) caused significant suppression of CNV, but with an associated weight loss suggestive of systemic toxicity. To minimize systemic exposure, topical delivery of TG100572 to the cornea was explored, and while substantial levels of TG100572 were achieved in the retina and choroid, superior exposure levels were achieved using TG100801, an inactive prodrug that generates TG100572 by de-esterification. Neither TG100801 nor TG100572 were detectable in plasma following topical delivery of TG100801, and adverse safety signals (such as weight loss) were not observed even with prolonged dosing schedules. Topical TG100801 significantly suppressed laser-induced CNV in mice, and reduced fluorescein leakage from the vasculature and retinal thickening measured by optical coherence tomography in a rat model of retinal vein occlusion. These data suggest that TG100801 may provide a new topically applied treatment approach for ocular neovascularization and retinal edema.
Journal of Cellular Physiology 08/2008; 216(1):29-37. · 3.87 Impact Factor
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Moorthy S S Palanki, Hideo Akiyama,
Peter Campochiaro,
Jianguo Cao,
Chun P Chow,
Luis Dellamary,
John Doukas,
Richard Fine,
Colleen Gritzen,
John D Hood, [......],
Andrew McPherson,
Ved P Pathak,
Joel Renick,
Richard Soll,
Naoyasu Umeda,
Shiyin Yee,
Katsutoshi Yokoi,
Binqi Zeng,
Hong Zhu,
Glenn Noronha
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ABSTRACT: Age-related macular degeneration (AMD) is one of the leading causes of loss of vision in the industrialized world. Attenuating the VEGF signal in the eye to treat AMD has been validated clinically. A large body of evidence suggests that inhibitors targeting the VEGFr pathway may be effective for the treatment of AMD. Recent studies using Src/YES knockout mice suggest that along with VEGF, Src and YES play a crucial role in vascular leak and might be useful in treating edema associated with AMD. Therefore, we have developed several potent benzotriazine inhibitors designed to target VEGFr2, Src, and YES. One of the most potent compounds is 4-chloro-3-{5-methyl-3-[4-(2-pyrrolidin-1-yl-ethoxy)phenylamino]benzo[1,2,4]triazin-7-yl}phenol ( 5), a dual inhibitor of both VEGFr2 and the Src family (Src and YES) kinases. Several ester analogues of 5 were prepared as prodrugs to improve the concentration of 5 at the back of the eye after topical administration. The thermal stability of these esters was studied, and it was found that benzoyl and substituted benzoyl esters of 5 showed good thermal stability. The hydrolysis rates of these prodrugs were studied to analyze their ability to undergo conversion to 5 in vivo so that appropriate concentrations of 5 are available in the back-of-the-eye tissues. From these studies, we identified 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate ( 12), a topically administered prodrug delivered as an eye drop that is readily converted to the active compound 5 in the eye. This topically delivered compound exhibited excellent ocular pharmacokinetics and poor systemic circulation and showed good efficacy in the laser induced choroidal neovascularization model. On the basis of its superior profile, compound 12 was advanced. It is currently in a clinical trial as a first in class, VEGFr2 targeting, topically applied compound for the treatment of AMD.
Journal of Medicinal Chemistry 04/2008; 51(6):1546-59. · 5.25 Impact Factor
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Raquel Lima e Silva,
Jikui Shen,
Sean F Hackett,
Shu Kachi, Hideo Akiyama,
Katsuji Kiuchi,
Katsutoshi Yokoi,
Maria C Hatara,
Thomas Lauer,
Sadia Aslam,
Yuan Yuan Gong,
Wei-Hong Xiao,
Naw Htee Khu,
Catherine Thut,
Peter A Campochiaro
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ABSTRACT: Hypoxia causes increased expression of several proteins that have the potential to promote neovascularization. Vascular endothelial growth factor (VEGF) is up-regulated by hypoxia in the retina and plays a central role in the development of several types of ocular neovascularization, but the effects of other hypoxia-regulated proteins are less clear. Stromal-derived factor-1 (SDF-1) and its receptor, CXCR4, have hypoxia response elements in the promoter regions of their genes and are increased in hypoxic liver and heart. In this study, we found that SDF-1 and CXCR4 are increased in hypoxic retina, with SDF-1 localized in glial cells primarily near the surface of the retina and CXCR4 localized in bone marrow-derived cells. Glial cells also expressed CXCR4, which suggested the possibility of autocrine stimulation, but influx of bone marrow-derived cells is the major source of increased levels of CXCR4. High levels of VEGF in the retina in the absence of hypoxia also increased levels of Cxcr4 and Sdf1 mRNA. CXCR4 antagonists reduced influx of bone marrow-derived cells into ischemic retina and strongly suppressed retinal neovascularization, VEGF-induced subretinal neovascularization, and choroidal neovascularization. These data suggest that SDF-1 and CXCR4 contribute to the involvement of bone marrow-derived cells and collaborate with VEGF in the development of several types of ocular neovascularization. They provide new targets for therapeutic intervention that may help to bolster and supplement effects obtained with VEGF antagonists.
The FASEB Journal 11/2007; 21(12):3219-30. · 5.71 Impact Factor
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Raquel Lima e Silva,
Shu Kachi, Hideo Akiyama,
JiKui Shen,
Maria Christina Hatara,
Sadia Aslam,
Yuan Yuan Gong,
Naw Htee Khu,
Thomas W Lauer,
Sean F Hackett,
Laurence J Marton,
Peter A Campochiaro
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ABSTRACT: Periocular injections of the polyamine analog CGC-11144 three times a week causes regression of choroidal neovascularization. This regimen was selected to maximize chances of success for proof of concept, but is not ideal for clinical application. In this study we explored other regimens for periocular delivery of CGC-11144, and 2 other polyamine analogs, CGC-11047 and CGC-11093. A single periocular injection of 200 microg of CGC-11144, 2 mg of CGC-11047, or 1.5 mg of CGC-11093 caused significant suppression and regression of laser-induced choroidal neovascularization. An injection of 2 mg of CGC-11047 or 1.5 mg of CGC-11093 one or two weeks before, but not 3 weeks before, rupture of Bruch's membrane also caused significant suppression. Periocular injection of polyamine analogs also caused strong inhibition of retinal or subretinal neovascularization in mice with oxygen-induced ischemic retinopathy or Rhodopsin promoter/VEGF transgenic mice, respectively. These data suggest that periocular injection of one of 3 different polyamine analogs inhibits retinal or choroidal neovascularization and a single injection provides inhibitory activity for at least 2 to 3 weeks, which could provide the basis for a feasible treatment regimen for clinical trials.
Experimental Eye Research 12/2006; 83(5):1260-7. · 3.26 Impact Factor
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ABSTRACT: Bolstering the endogenous oxidative damage defense system is a good strategy for development of treatments to combat neurodegenerative diseases in which oxidative damage plays a role. A first step in such treatment development is to determine the role of various components of the defense system in cells that degenerate. In this study, we sought to determine the role of superoxide dismutase 1 (SOD1) in two models of oxidative damage-induced retinal degeneration. In one model, paraquat is injected into the vitreous cavity and then enters retinal cells and generates reactive oxygen species (ROS) that cause progressive retinal damage. Assessment of retinal function with serial electroretinograms (ERGs) showed that sod1 -/- mice were much more sensitive than sod1 +/+ mice to the damaging effects of paraquat, while sod1 +/- mice showed intermediate sensitivity. Compared to sod1 +/+ mice, sod1 -/- mice showed greater paraquat-induced oxidative damage and apoptosis. In the second model, mice were exposed to hyperoxia for several weeks, and sod1 -/- mice showed significantly greater reductions in ERG amplitudes than sod1 +/+ mice. In both of these models, transgenic mice carrying a sod1 transgene driven by a beta-actin promoter showed less oxidative stress-induced reduction in ERG amplitudes. These data demonstrate that SOD1 protects retinal cells against paraquat- and hyperoxia-induced oxidative damage and suggest that overexpression of SOD1 should be considered as one component of ocular gene therapy to prevent oxidative damage-induced retinal degeneration.
Journal of Cellular Physiology 10/2006; 208(3):516-26. · 3.87 Impact Factor
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Raquel Lima E Silva,
Shu Kachi, Hideo Akiyama,
Jikui Shen,
Sadia Aslam,
Yuan Yuan Gong,
Naw Htee Khu,
Maria C Hatara,
Ariel Boutaud,
Robert Peterson,
Peter A Campochiaro
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ABSTRACT: Vascular endothelial cells receive proangiogenic or antiangiogenic signals from components of extracellular matrix (ECM) depending upon the situation and many molecular signals can have opposite effects in different vascular beds. Tissue inhibitor of metalloproteinase 1 is antiangiogenic in several tissues, but promotes retinal neovascularization. When cleaved from native collagens, several of the non-collagenous domains (NC1) of basement membrane collagens have antiangiogenic effects in some tissues, but this is context dependent for the NC1 of the alpha 1 chain of collagen IV. It is critical to examine effects in several well-defined model systems before assuming that an ECM component is universally antiangiogenic. In this study, we examined the effects of a recombinant fragment of NC1 of the alpha 2 chain of type IV collagen (alpha2(IV)NC1) in a well-characterized model of ocular neovascularization. Intravitreous or periocular injections of alpha2(IV)NC1 caused selective apoptosis of endothelial cells participating in neovascularization resulting in suppression of neovascularization when the peptide was given prior to onset of new vessel sprouting. Importantly, when the peptide was given after neovascularization had already developed, it caused the new vessels to regress. This suggests that alpha2(IV)NC1, which has previously been shown to suppress tumor angiogenesis in xenograft models, is also a strong antiangiogenic agent in the choroid and is a therapeutic candidate for treatment of neovascular age-related macular degeneration.
Journal of Cellular Physiology 08/2006; 208(1):161-6. · 3.87 Impact Factor
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ABSTRACT: Integrin alpha(5)beta(1) plays an important role in developmental angiogenesis, but its role in various types of pathologic neovascularization has not been completely defined. In this study, we found strong up-regulation of alpha(5)beta(1) in choroidal neovascularization. Implantation of an osmotic pump delivering 1.5 or 10 microg/h ( approximately 1.8 or 12 mg/kg/day) of 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidin-3-yloxy}-acetylamino)-2-(alkylamino)-propionic acid (JSM6427), a selective alpha(5)beta(1) antagonist, caused significant suppression of choroidal neovascularization; the area of neovascularization was reduced by 33 to 40%. When an osmotic pump delivering 10 microg/h of JSM6427 was implanted 7 days after rupture of Bruch's membrane, there was terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in vascular cells within the neovascularization and significant regression of the neovascularization over the next week. JSM6427 also induced apoptosis of cultured vascular endothelial cells. Fibronectin stimulates phosphorylation of extracellular signal-regulated kinase (ERK) in alpha(5)beta(1)-expressing cells that is blocked by JSM6427. These data suggest that alpha(5)beta(1) plays a role in the development and maintenance of choroidal neovascularization and provides a target for therapeutic intervention.
Molecular Pharmacology 07/2006; 69(6):1820-8. · 4.88 Impact Factor
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ABSTRACT: Platelet-derived growth factor-B (PDGF-B) has been implicated in the pathogenesis of proliferative retinopathies and other scarring disorders in the eye. In this study, we sought to test the therapeutic potential of an aptamer that selectively binds PDGF-B, ARC126, and its PEGylated derivative, ARC127. Both ARC126 and ARC127 blocked PDGF-B-induced proliferation of cultured fibroblasts with an IC50 of 4 nM. Pharmacokinetic studies in rabbits showed similar peak vitreous concentrations of approximately 110 microM after intravitreous injection of 1 mg of either ARC126 or ARC127, but the terminal half-life was longer for ARC127 (98 versus 43 h). Efficacy was tested in rho/PDGF-B transgenic mice that express PDGF-B in photoreceptors and develop severe proliferative retinopathy resulting in retinal detachment. Compared to eyes injected with 20 microg of scrambled aptamer in which five of six developed detachments (three total and two partial), eyes injected with ARC126 (no detachment in five of six and one partial detachment), or ARC127 (no detachment in six of six) had significantly fewer retinal detachments. They also showed a significant reduction in epiretinal membrane formation. These data demonstrate that a single intravitreous injection of an aptamer that specifically binds PDGF-B is able to significantly reduce epiretinal membrane formation and retinal detachment in rho/PDGF-B mice. These striking effects in an aggressive model of proliferative retinopathy suggest that ARC126 and ARC127 should be considered for treatment of diseases in which PDGF-B has been implicated, including ischemic retinopathies such as proliferative diabetic retinopathy, proliferative vitreoretinopathy (PVR), and choroidal neovascularization.
Journal of Cellular Physiology 06/2006; 207(2):407-12. · 3.87 Impact Factor
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Hideo Akiyama,
Khalid A Mohamedali,
Raquel Lima E Silva,
Shu Kachi,
Jikui Shen,
Christina Hatara,
Naoyasu Umeda,
Sean F Hackett,
Sadia Aslam,
Melissa Krause,
Hong Lai,
Michael G Rosenblum,
Peter A Campochiaro
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ABSTRACT: Tumors provide an extremely abnormal microenvironment that stimulates neovascularization from surrounding vessels and causes altered gene expression within vascular cells. Up-regulation of vascular endothelial growth factor (VEGF) receptors has allowed selective destruction of tumor vessels by administration of a chimeric protein consisting of VEGF121 coupled to the toxin gelonin (VEGF/rGel). We sought to determine whether there is sufficient up-regulation of VEGF receptors in endothelial cells participating in ocular neovascularization to permit a similar strategy. After intravenous injection of 45 mg/kg VEGF/rGel, but not uncoupled recombinant gelonin (rGel), there was immunofluorescent staining for rGel within choroidal neovascularization in mice and regression of the neovascularization occurred, demonstrating successful vascular targeting via the systemic circulation. Intraocular injection of 5 ng of VEGF/rGel also caused significant regression of choroidal neovascularization and regression of retinal neovascularization in two models, transgenic mice with expression of VEGF in photoreceptors and mice with ischemic retinopathy, whereas injection of 5 ng of rGel had no effect. These data suggest that the strategy of vascular targeting can be applied to nonmalignant neovascular diseases and could serve as the basis of a new treatment to reduce established ocular neovascularization.
Molecular Pharmacology 01/2006; 68(6):1543-50. · 4.88 Impact Factor
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Raquel Lima e Silva,
Yoshitsugu Saishin,
Yumiko Saishin, Hideo Akiyama,
Shu Kachi,
Sadia Aslam,
Brian Rogers,
Tye Deering,
Yuan Yuan Gong,
Sean F Hackett,
Hong Lai,
Benjamin J Frydman,
Aldonia Valasinas,
Laurence J Marton,
Peter A Campochiaro
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ABSTRACT: Polyamine analogues inhibit tumor growth in vitro and in vivo, and oligoamines with a chain length of 10, 12, or 14 are particularly potent. This study was conducted to investigate the effect of the decamines CGC-11144 and CGC-11150 in a mouse model of choroidal neovascularization (CNV).
Mice with laser-induced rupture of Bruch's membrane were given intraperitoneal, intravitreous, or periocular injection of CGC-11144, CGC-11150, or vehicle, and after 14 days, they were perfused with fluorescein-labeled dextran, and the area of CNV was measured on choroidal flatmounts by image analysis. In some groups of mice, treatments were started 7 days after rupture of Bruch's membrane to determine the effect of the agent on established CNV. Electroretinograms (ERGs) were performed to assess the effects on retinal function, and histopathology was used to evaluate retinal structure.
Intraperitoneal injection of 10 or 20 mg/kg CGC-11144 or CGC-11150 resulted in small but significant reductions in the area of CNV. Intravitreous injection of 20 microg CGC-11144 or CGC-11150 on days 0 and 7 after rupture of Bruch's membrane resulted in a approximately 40% reduction in the area of CNV, with a similar reduction after periocular injections of 0.2 mg CGC-11144 three times a week for 2 weeks. Both intravitreous and periocular delivery of CGC-11144 also caused significant regression of established CNV. Within 2 days of periocular injection of CGC-11144, there was apoptosis in CNV lesions, but not in normal blood vessels or other retinal cells. Periocular injections of d,l-alpha-difluoromethyl-ornithine (DFMO), which decreases polyamine levels by a different mechanism, also inhibited CNV. There was no decline in ERG amplitudes or abnormal retinal morphology after daily injections of 0.2 mg CGC-11144 for 2 weeks, but a single intravitreous injection compromised retinal structure and function.
Periocular delivery of the polyamine analogues may be a useful approach for the treatment of CNV.
Investigative Ophthalmology & Visual Science 10/2005; 46(9):3323-30. · 3.60 Impact Factor
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ABSTRACT: Vascular endothelial growth factor (VEGF) plays a central role in vasoproliferative diseases in the retina, however, other gene products modulate its effects. The angiopoietins are particularly important in this regard. Angiopoietin 2 (Ang2) collaborates with VEGF to stimulate neovascularization (NV) in some situations, but in other situations causes regression of NV. Ang2 also causes a transient increase in vascular density during retinal vascular development. In this study, we sought to determine if Ang1 has similar activities. The effects of Ang1 were tested in double transgenic mice with inducible expression of Ang1. Increased expression of Ang1 in the retina during retinal vascular development did not cause a detectable alteration in vascular density. Also, unlike Ang2, increased expression of Ang1 had no effect on established retinal or choroidal NV. However, when Ang1 expression was initiated simultaneously with that of VEGF, it strongly suppressed VEGF-induced NV and prevented retinal detachment. These data indicate that the timing of Ang1 expression is a critical determinate of its effects on VEGF-induced NV in the retina; it effectively blocks the initiation and progression of NV, but cannot reverse established NV or reduce leakage from NV. These data suggest that increased expression of Ang1 may be a good strategy for prophylaxis of retinal NV, but is unlikely to be effective as monotherapy of established NV.
Journal of Cellular Physiology 08/2005; 204(1):227-35. · 3.87 Impact Factor
