Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 19 de 19
Filter
Add more filters










Publication year range
1.
PLoS One ; 8(8): e71808, 2013.
Article in English | MEDLINE | ID: mdl-23977149

ABSTRACT

Although blocking VEGF has a positive effect in wet age-related macular degeneration (AMD), the effect of blocking its receptors remains unclear. This was an investigation of the effect of VEGF receptor (VEGFR) 1 and/or 2 blockade on retinal microglia/macrophage infiltration in laser-induced choroidal neovascularization (CNV), a model of wet AMD. CNV lesions were isolated by laser capture microdissection at 3, 7, and 14 days after laser and analyzed by RT-PCR and immunofluorescence staining for mRNA and protein expression, respectively. Neutralizing antibodies for VEGFR1 or R2 and the microglia inhibitor minocycline were injected intraperitoneally (IP). Anti-CD11b, CD45 and Iba1 antibodies were used to confirm the cell identity of retinal microglia/macrophage, in the RPE/choroidal flat mounts or retinal cross sections. CD11b(+), CD45(+) or Iba1(+) cells were counted. mRNA of VEGFR1 and its three ligands, PlGF, VEGF-A (VEGF) and VEGF-B, were expressed at all stages, but VEGFR2 were detected only in the late stage. PlGF and VEGF proteins were expressed at 3 and 7 days after laser. Anti-VEGFR1 (MF1) delivered IP 3 days after laser inhibited infiltration of leukocyte populations, largely retinal microglia/macrophage to CNV, while anti-VEGFR2 (DC101) had no effect. At 14 days after laser, both MF1 and DC101 antibodies markedly inhibited retinal microglia/macrophage infiltration into CNV. Therefore, VEGFR1 and R2 play differential roles in the pathogenesis of CNV: VEGFR1 plays a dominant role at 3 days after laser; but both receptors play pivotal roles at 14 days after laser. In vivo imaging demonstrated accumulation of GFP-expressing microglia into CNV in both CX3CR1(gfp/gfp) and CX3CR1(gfp/+) mice. Minocycline treatment caused a significant increase in lectin(+) cells in the sub-retinal space anterior to CNV and a decrease in dextran-perfused neovessels compared to controls. Targeting the chemoattractant molecules that regulate trafficking of retinal microglia/macrophage appears to be a compelling therapeutic strategy to control CNV and treat wet AMD.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/administration & dosage , Choroidal Neovascularization/drug therapy , Macrophages/immunology , Microglia/immunology , Retina/immunology , Vascular Endothelial Growth Factor Receptor-1/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Animals , Cell Movement , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Disease Models, Animal , Humans , Injections, Intraperitoneal , Laser Capture Microdissection , Lasers , Macrophages/drug effects , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/drug effects , Minocycline/administration & dosage , Retina/drug effects , Tissue Culture Techniques , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Wet Macular Degeneration/drug therapy , Wet Macular Degeneration/metabolism , Wet Macular Degeneration/pathology
2.
Invest Ophthalmol Vis Sci ; 52(10): 7565-73, 2011 Sep 29.
Article in English | MEDLINE | ID: mdl-21873682

ABSTRACT

PURPOSE: Prolyl hydroxylases (PHDs) are oxygen sensors that stabilize hypoxia-inducible factors (HIFs) to induce proinflammatory, vasopermeability, and proapoptotic factors. These may be potential targets to reduce the complications of ischemic retinopathies. METHODS: Oxygen-induced ischemic retinopathy (OIR) was generated as a model for retinopathy of prematurity (ROP) by placing 7-day-old mice in 75% oxygen for 5 days and returning them to the relative hypoxia of room air for 5 days. Neovascularization (NV) and avascular areas were assessed on retinal flat-mounts by image analysis. Blood-retinal barrier breakdown was assessed using ³H-mannitol as a tracer. Apoptosis was detected with TUNEL staining. HIF-1α and VEGF were quantified using Western blot analysis and ELISA. RESULTS: PHD1-deficient mice demonstrated reduced hyperoxia-associated vascular obliteration during oxygen-induced ischemic retinopathy. This was associated with subsequent reduced avascularity, vascular leakage, and pathologic NV during the hypoxic phase, which could be accounted for by a reduced expression of HIF-1α and VEGF. Apoptosis in the retina was also reduced in PHD1-depleted mice after 2 days in hyperoxia. CONCLUSIONS: PHD1 deficiency is associated with a reduction of ischemia-induced retinal NV. The regulatory mechanism in this model appears to be: PHD1 depletion prevents HIF-1α degradation in hyperoxia, which induces VEGF, thus preventing hyperoxia-related vessel loss. Without a vessel deficiency, there would not be relative hypoxia when the mice are returned to room air and there would be no need to initiate angiogenesis signaling. Blocking PHD1 may be beneficial for ischemic retinopathies and inflammatory and neurodegenerative disorders.


Subject(s)
Procollagen-Proline Dioxygenase/deficiency , Retinal Neovascularization/prevention & control , Animals , Animals, Newborn , Apoptosis , Blood-Retinal Barrier , Blotting, Western , Capillary Permeability , Enzyme-Linked Immunosorbent Assay , Erythropoietin/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , In Situ Nick-End Labeling , Infant, Newborn , Mice , Mice, Knockout , Oxygen/toxicity , Reperfusion Injury , Retinal Neovascularization/enzymology , Retinal Vessels , Retinopathy of Prematurity , Vascular Endothelial Growth Factor A/metabolism
3.
PLoS One ; 6(6): e21411, 2011.
Article in English | MEDLINE | ID: mdl-21731737

ABSTRACT

OBJECTIVE: VEGFR1 and 2 signaling have both been increasingly shown to mediate complications of ischemic retinopathies, including retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and diabetic retinopathy (DR). This study evaluates the effects of blocking VEGFR1 and 2 on pathological angiogenesis and vascular leakage in ischemic retinopathy in a model of ROP and in choroidal neovascularization (CNV) in a model of AMD. MATERIALS AND METHODS: Neutralizing antibodies specific for mouse VEGFR1 (MF1) and VEGFR2 (DC101) were administrated systemically. CNV was induced by laser photocoagulation and assessed 14d after laser treatment. Retinal NV was generated in oxygen-induced ischemic retinopathy (OIR) and assessed at p17. NV quantification was determined by measuring NV tufts and vascular leakage was quantified by measuring [(3)H]-mannitol leakage from blood vessels into the retina. Gene expression was measured by real-time quantitative (Q)PCR. RESULTS: VEGFR1 and VEGFR2 expressions were up-regulated during CNV pathogenesis. Both MF1 and DC101 significantly suppressed CNV at 50 mg/kg: DC101 suppressed CNV by 73±5% (p<0.0001) and MF1 by 64±6% (p = 0.0002) in a dosage-dependent manner. The combination of MF1 and DC101 enhanced the inhibitory efficacy and resulted in an accumulation of retinal microglia at the CNV lesion. Similarly, both MF1 and DC101 significantly suppressed retinal NV in OIR at 50 mg/kg: DC101 suppressed retinal NV by 54±8% (p = 0.013) and MF1 by 50±7% (p<0.0002). MF1 was even more effective at inhibiting ischemia-induced BRB breakdown than DC101: the retina/lung leakage ratio for MF1 was reduced by 73±24%, p = 0.001 and for DC101 by 12±4%, p = 0.003. The retina/renal leakage ratio for MF1 was reduced by 52±28%, p = 0.009 and for DC101 by 13±4%, p = 0.001. CONCLUSION: Our study provides further evidence that both VEGFR1 and 2 mediate pathological angiogenesis and vascular leakage in these models of ocular disease and suggests that antagonist antibodies to these receptor tyrosine kinases (RTKs) are potential therapeutic agents.


Subject(s)
Blood Vessels/pathology , Choroidal Neovascularization/pathology , Eye/blood supply , Eye/pathology , Vascular Endothelial Growth Factor Receptor-1/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Angiogenesis Inducing Agents/metabolism , Animals , Choroidal Neovascularization/complications , Choroidal Neovascularization/etiology , Inflammation/complications , Inflammation/pathology , Ischemia/complications , Ischemia/pathology , Lectins/metabolism , Ligands , Mice , Microglia/pathology , Retina/pathology , Retinal Diseases/complications , Retinal Diseases/metabolism , Retinal Diseases/pathology , Up-Regulation , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism
4.
Curr Eye Res ; 36(8): 747-53, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21780924

ABSTRACT

PURPOSE: The following study was designed to investigate early biosynthetic and ultrastructural changes that alter functional properties of the basement membrane (BM) and affect vascular permeability in diabetic retinopathy. MATERIALS AND METHODS: To determine whether altered matrix synthesis affects cell monolayer permeability, rat retinal endothelial cells (RRECs) were grown for 4 days to confluency in normal (N, 5 mM) or high glucose (HG, 30 mM) medium on transwell inserts and subjected to an in vitro cell monolayer permeability assay. Inserts were cut out and viewed under a transmission electron microscope to assess extracellular matrix (ECM) accumulation and cell morphology. In parallel cell cultures, fibronectin and collagen IV protein expression were determined using Western Blot analysis. RESULTS: Electron microscopic analysis of cells exposed to short-term HG showed no difference in inter-endothelial cell tight junctions (TJs) or in the number of vesicles or coated pits compared to those of normal cells. However, ECM accumulation underlying HG cells was significantly increased compared to that of cells grown in N medium (139 ± 7% of control, p = 0.04), with areas of focal thickening. Western blot analysis showed increased fibronectin and collagen IV expression (152 ± 24% of control, p = 0.01; 146 ± 16% of control, p = 0.02, respectively) in cells grown in HG compared to those grown in N medium. Cell monolayers grown in HG exhibited increased permeability to FITC-dextran compared to cells grown in N medium (134 ± 15% of control, p = 0.02). CONCLUSIONS: High glucose-induced excess ECM accumulation and altered composition underlies structural and functional changes that allow increased permeability. This finding provides evidence for the first time that the thickened vascular basement membrane contributes to the development of excess permeability seen in diabetic retinopathy.


Subject(s)
Basement Membrane/drug effects , Capillary Permeability/drug effects , Diabetic Retinopathy/metabolism , Endothelium, Vascular/drug effects , Extracellular Matrix/metabolism , Glucose/pharmacology , Sweetening Agents/pharmacology , Animals , Basement Membrane/metabolism , Basement Membrane/ultrastructure , Blotting, Western , Cells, Cultured , Collagen Type IV/metabolism , Dextrans/metabolism , Diabetic Retinopathy/pathology , Endothelium, Vascular/metabolism , Extracellular Matrix/ultrastructure , Fibronectins/metabolism , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/metabolism , Rats , Retinal Vessels/cytology , Tight Junctions , Up-Regulation
5.
PLoS One ; 6(3): e18076, 2011 Mar 25.
Article in English | MEDLINE | ID: mdl-21464949

ABSTRACT

Increased vascular permeability is an early event characteristic of tissue ischemia and angiogenesis. Although VEGF family members are potent promoters of endothelial permeability the role of placental growth factor (PlGF) is hotly debated. Here we investigated PlGF isoforms 1 and 2 and present in vitro and in vivo evidence that PlGF-1, but not PlGF-2, can inhibit VEGF-induced permeability but only during a critical window post-VEGF exposure. PlGF-1 promotes VE-cadherin expression via the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter and subsequently stabilizes transendothelial junctions, but only after activation of endothelial cells by VEGF. PlGF-1 regulates vascular permeability associated with the rapid localization of VE-cadherin to the plasma membrane and dephosphorylation of tyrosine residues that precedes changes observed in claudin 5 tyrosine phosphorylation and membrane localization. The critical window during which PlGF-1 exerts its effect on VEGF-induced permeability highlights the importance of the translational significance of this work in that PLGF-1 likely serves as an endogenous anti-permeability factor whose effectiveness is limited to a precise time point following vascular injury. Clinical approaches that would pattern nature's approach would thus limit treatments to precise intervals following injury and bring attention to use of agents only during therapeutic windows.


Subject(s)
Adherens Junctions/drug effects , Adherens Junctions/metabolism , Capillary Permeability/drug effects , Pregnancy Proteins/pharmacology , Vascular Endothelial Growth Factor A/pharmacology , Animals , Antigens, CD/genetics , Base Sequence , Cadherins/genetics , Claudins/metabolism , Humans , Mice , Molecular Sequence Data , Phosphorylation/drug effects , Placenta Growth Factor , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor/metabolism , Tight Junctions/drug effects , Tight Junctions/metabolism , Time Factors , Transcriptional Activation/drug effects , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism
6.
Mol Vis ; 17: 492-507, 2011 Feb 17.
Article in English | MEDLINE | ID: mdl-21364963

ABSTRACT

PURPOSE: The role of vascular endothelial growth factor (VEGF)-B in the eye is poorly understood. The present study was conducted to evaluate the effect of overexpression of VEGF-B via adeno-associated virus (AAV) gene transfer on ocular angiogenesis, inflammation, and the blood-retinal barrier (BRB). METHODS: Three recombinant AAV vectors were prepared, expressing the 167 (AAV-VEGF-B167) or 186 amino acid isoform (AAV-VEGF-B186) of VEGF-B or the green fluorescent protein (GFP) reporter gene (AAV-GFP). Approximately 1 x 109 viral genome copies of AAV-VEGF-B167, AAV-VEGF-B186, or AAV-GFP were intraocularly injected. The efficacy of the gene transfer was assessed by directly observing GFP, by immunohistochemistry, or by real-time PCR. A leukostasis assay using fluorescein isothiocyanate-conjugated Concanavalin A was used to evaluate inflammation. The BRB was assessed using a quantitative assay with ³H-mannitol as a tracer. Retinal neovascularization (NV) was assessed at postnatal day 17 in oxygen-induced ischemic retinopathy after intravitreal injection of AAV-VEGF-B in left eyes and AAV-GFP in right eyes at postnatal day 7. Two weeks after injection of AAV vectors, choroidal NV was generated by laser photocoagulation and assessed 2 weeks later. RESULTS: GFP expression was clearly demonstrated, primarily in the retinal pigment epithelium (RPE) and outer retina, 1-6 weeks after delivery. mRNA expression levels of VEGF-B167 and VEGF-B186 were 5.8 and 12 fold higher in the AAV-VEGF-B167- and AAV-VEGF-B186-treated groups, respectively. There was no evidence of an inflammatory response or vessel abnormality following injection of the vectors in normal mice; however, VEGF-B increased retinal and choroidal neovascularization. AAV-VEGF-B186, but not AAV-VEGF-B167, enhanced retinal vascular permeability. CONCLUSIONS: VEGF-B overexpression promoted pathological retinal and choroidal NV and BRB breakdown without causing inflammation, which is associated with the progression of diabetic retinopathy and age-related macular degeneration, showing that these complications are not dependent on inflammation. VEGF-B targeting could benefit antiangiogenic therapy.


Subject(s)
Capillary Permeability/physiology , Choroidal Neovascularization/physiopathology , Gene Transfer Techniques , Inflammation/complications , Retinal Neovascularization/physiopathology , Vascular Endothelial Growth Factor B/genetics , Animals , Choroidal Neovascularization/complications , Choroidal Neovascularization/genetics , Dependovirus/genetics , Genetic Vectors/genetics , Green Fluorescent Proteins/metabolism , Inflammation/physiopathology , Ischemia/complications , Ischemia/physiopathology , Mice , Mice, Inbred C57BL , Oxygen , Recombination, Genetic/genetics , Retina/pathology , Retina/physiopathology , Retinal Neovascularization/complications , Retinal Neovascularization/genetics , Transgenes/genetics
7.
Invest Ophthalmol Vis Sci ; 52(3): 1336-44, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21212173

ABSTRACT

PURPOSE: Blood-retinal barrier [BRB] breakdown, characteristic of diabetic retinopathy (DR), is believed to depend on inflammation and apoptosis. Retinal inflammation is almost completely suppressed in the absence of TNFα, which is also associated with apoptosis. This study was conducted to determine the role of TNFα in these diabetic complications. METHODS: Diabetes was induced with streptozotocin in Tnfa knockout (KO) mice, to provide a chemical model of diabetes, and Tnfa (KO) mice were crossed with Ins2(Akita) mice to generate a genetic model, with both models being devoid of TNFα. The BRB was assessed at 1, 1.5, 3, and 6 months. Leukostasis was assessed using FITC-conjugated ConA to label leukocytes. Apoptosis was assessed with TUNEL and activated caspase-3 staining. PECAM1 identified endothelial cells, and SMA identified pericytes. RESULTS: At 1 month of diabetes, the absence of TNFα had no effect on DR-associated BRB breakdown, even though it prevented retinal leukostasis, demonstrating that neither TNFα nor inflammation is essential for early BRB breakdown in DR in either model of diabetes. At 3 months of diabetes, BRB breakdown was significantly suppressed and at 6 months, it was completely prevented in the absence of TNFα in both models, showing that TNFα is essential for progressive BRB breakdown. DR-mediated apoptosis in the retina, which appears to involve endothelial cells, pericytes, and neurons, was inhibited in the absence of TNFα in both models. CONCLUSIONS: Although neither TNFα nor inflammation is necessary for early BRB breakdown in DR, TNFα is critical for later complications and would be a good therapeutic target for the prevention of the progressive BRB breakdown, retinal leukostasis, and apoptosis associated with DR.


Subject(s)
Apoptosis , Blood-Retinal Barrier/metabolism , Diabetic Retinopathy/metabolism , Leukostasis/prevention & control , Retinal Neurons/pathology , Retinal Vessels/pathology , Tumor Necrosis Factor-alpha/physiology , Actins/metabolism , Animals , Capillary Permeability/physiology , Caspase 3/metabolism , Cell Survival , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetic Retinopathy/pathology , Enzyme-Linked Immunosorbent Assay , Female , Fluorescent Antibody Technique, Indirect , Gene Silencing/physiology , Genotype , In Situ Nick-End Labeling , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Reverse Transcriptase Polymerase Chain Reaction
8.
Invest Ophthalmol Vis Sci ; 51(11): 5825-32, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20538980

ABSTRACT

PURPOSE: Parstatin is a 41-mer peptide formed by proteolytic cleavage on activation of the PAR1 receptor. The authors recently showed that parstatin is a potent inhibitor of angiogenesis. The purpose of the present study was to evaluate the therapeutic effect of parstatin on ocular neovascularization. METHODS: Choroidal neovascularization was generated in mice using laser-induced rupture of Bruch's membrane and was assessed after 14 days after perfusion of FITC-dextran. Oxygen-induced retinal neovascularization was established in neonatal mice by exposing them to 75% O(2) at postnatal day (P)7 for 5 days and then placing them in room air for 5 days. Evaluation was performed on P17 after staining with anti-mouse PECAM-1. The effect of parstatin was tested after intravitreal administration. The effects of subconjunctival-injected parstatin on corneal neovascularization and inflammation in rats were assessed 7 days after chemical burn-induced corneal neovascularization. Retinal leukostasis in mice was assessed after perfusion with FITC-conjugated concanavalin A. RESULTS: Parstatin potently inhibited choroidal neovascularization with an IC(50) of approximately 3 µg and a maximum inhibition of 59% at 10 µg. Parstatin suppressed retinal neovascularization with maximum inhibition of 60% at 3 µg. Ten-microgram and 30-µg doses appeared to be toxic to the neonatal retina. Subconjunctival parstatin inhibited corneal neovascularization, with 200 µg the most effective dose (59% inhibition). In addition, parstatin significantly inhibited corneal inflammation and VEGF-induced retinal leukostasis. In all models tested, scrambled parstatin was without any significant effect. CONCLUSIONS: Parstatin is a potent antiangiogenic agent of ocular neovascularization and may have clinical potential in the treatment of angiogenesis-related ocular disorders.


Subject(s)
Choroidal Neovascularization/prevention & control , Corneal Neovascularization/prevention & control , Disease Models, Animal , Keratitis/prevention & control , Peptide Fragments/therapeutic use , Receptor, PAR-1/therapeutic use , Animals , Choroidal Neovascularization/pathology , Conjunctiva/drug effects , Corneal Neovascularization/pathology , Intravitreal Injections , Keratitis/pathology , Leukostasis/prevention & control , Male , Mice , Mice, Inbred C57BL , Rats , Rats, Sprague-Dawley , Retinal Diseases/prevention & control , Retinal Neovascularization/pathology , Retinal Neovascularization/prevention & control
9.
Cell ; 141(1): 178-90, 2010 Apr 02.
Article in English | MEDLINE | ID: mdl-20371353

ABSTRACT

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.


Subject(s)
Neovascularization, Physiologic/drug effects , Pregnancy Proteins/antagonists & inhibitors , Pregnancy Proteins/metabolism , Angiogenesis Inhibitors/therapeutic use , Animals , Antibodies, Monoclonal/therapeutic use , Carcinoma, Hepatocellular/blood supply , Carcinoma, Hepatocellular/prevention & control , Choroid/blood supply , Disease Models, Animal , Eye Diseases/pathology , Humans , Liver Neoplasms, Experimental/blood supply , Liver Neoplasms, Experimental/prevention & control , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Papilloma/blood supply , Papilloma/chemically induced , Papilloma/prevention & control , Placenta Growth Factor , Skin Neoplasms/blood supply , Skin Neoplasms/chemically induced , Skin Neoplasms/prevention & control
10.
J Cell Physiol ; 217(1): 13-22, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18543272

ABSTRACT

Several ocular diseases complicated by neovascularization are being treated by repeated intraocular injections of vascular endothelial growth factor (VEGF) antagonists. While substantial benefits have been documented, there is concern that unrecognized damage may be occurring, because blockade of VEGF may damage the fenestrated vessels of the choroicapillaris and deprive retinal neurons of input from a survival factor. One report has suggested that even temporary blockade of all isoforms of VEGF-A results in significant loss of retinal ganglion cells. In this study, we utilized double transgenic mice with doxycycline-inducible expression of soluble VEGF receptor 1 coupled to an Fc fragment (sVEGFR1Fc), a potent antagonist of several VEGF family members, including VEGF-A, to test the effects of VEGF blockade in the retina. Expression of sVEGFR1Fc completely blocked VEGF-induced retinal vascular permeability and significantly suppressed the development of choroidal neovascularization at rupture sites in Bruch's membrane, but did not cause regression of established choroidal neovascularization. Mice with constant expression of sVEGFR1Fc in the retina for 7 months had normal electroretinograms and normal retinal and choroidal ultrastructure including normal fenestrations in the choroicapillaris. They also showed no significant difference from control mice in the number of ganglion cell axons in optic nerve cross sections and the retinal level of mRNA for 3 ganglion cell-specific genes. These data indicate that constant blockade of VEGF for up to 7 months has no identifiable deleterious effects on the retina or choroid and support the use of VEGF antagonists in the treatment of retinal diseases.


Subject(s)
Choroidal Neovascularization , Neovascularization, Physiologic , Retinal Ganglion Cells/ultrastructure , Retinal Neovascularization , Retinal Vessels/ultrastructure , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Apoptosis , In Situ Nick-End Labeling , Mice , Mice, Transgenic , Microscopy, Electron, Transmission , RNA, Messenger/analysis , Retinal Ganglion Cells/metabolism , Retinal Vessels/metabolism , Reverse Transcriptase Polymerase Chain Reaction
11.
J Neuroimmunol ; 182(1-2): 73-9, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17107717

ABSTRACT

Vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-alpha) show significant overlap with regard to their effects in the eye. It has been postulated that VEGF-induced leukostasis, breakdown of the blood-retinal barrier, and ischemia-induced retinal neovascularization may be mediated, at least in part, through TNF-alpha. In this study, we used mice deficient in TNF-alpha to test our hypothesis. Compared to wild type mice, TNF-alpha-deficient mice showed an 80% reduction in leukocyte accumulation in retinal vessels after intravitreous injection of VEGF, and 100% reductions after intravitreous injections of interleukin-1beta (IL-1beta) or platelet-activating factor (PAF). The increase in retinal vascular permeability induced by injection of PAF was significantly reduced in mice lacking TNF-alpha, but VEGF- and IL-1beta-induced leakage was unaffected. Compared to wild type mice with oxygen-induced ischemic retinopathy, TNF-alpha-deficient mice with ischemic retinopathy showed significantly reduced leukostasis and mild reduction in vascular leakage, but no significant difference in retinal neovascularization. These data suggest that TNF-alpha mediates VEGF-, IL-1beta-, and PAF-induced leukostasis and vascular leakage mediated by PAF, but not leakage caused by VEGF or IL-1beta. Ischemia-induced retinal neovascularization, which has previously been shown to require VEGF, does not require TNF-alpha and is unaffected by attenuation of leukostasis.


Subject(s)
Blood-Retinal Barrier , Ischemia/complications , Ischemia/metabolism , Leukostasis/etiology , Retinal Neovascularization/metabolism , Retinal Vessels , Tumor Necrosis Factor-alpha/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Blood-Retinal Barrier/drug effects , Capillary Permeability/drug effects , Interleukin-1beta/pharmacology , Ischemia/chemically induced , Leukostasis/chemically induced , Mice , Mice, Knockout , Oxygen , Platelet Activating Factor/pharmacology , Tumor Necrosis Factor-alpha/deficiency , Tumor Necrosis Factor-alpha/pharmacology , Vascular Endothelial Growth Factor A/pharmacology
12.
J Cell Physiol ; 206(3): 749-58, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16245301

ABSTRACT

Retinal neovascularization (NV) and macular edema, resulting from blood-retinal barrier (BRB) breakdown, are major causes of visual loss in ischemic retinopathies. Choroidal NV (CNV) occurs in diseases of the retinal pigmented epithelium/Bruch's membrane complex and is another extremely prevalent cause of visual loss. We used mice in which the hypoxia response element (HRE) is deleted from the vascular endothelial growth factor (vegf) promoter (Vegf(delta/delta) mice) to explore the role of induction of VEGF through the HRE in these disease processes. Compared to wild type (Vegf+/+) mice with oxygen-induced ischemic retinopathy (OIR) in which vegf mRNA levels were increased and prominent retinal NV and BRB breakdown occurred, Vegf(delta/delta) littermates with OIR failed to increase vegf mRNA levels in the retina and had significantly less retinal NV and BRB breakdown, but showed prominent dilation of some superficial retinal vessels. Vegf(+/delta) littermates with ischemic retinopathy developed comparable retinal NV to Vegf+/+ mice, exhibited intermediate levels of BRB breakdown, and did not show vasodilation. In a mouse model of CNV, due to laser-induced rupture of Bruch's membrane, the area of CNV at Bruch's membrane rupture sites was more than tenfold greater in Vegf+/+ mice than in Vegf(delta/delta) littermates. In contrast to these dramatic differences in pathologic ocular NV, Vegf(delta/delta) mice showed subtle differences in retinal vascular development compared to Vegf+/+ mice; it was slightly delayed, but otherwise normal. These data suggest that induction of VEGF through the HRE in its promoter is critical for retinal and CNV, but not for retinal vascular development.


Subject(s)
Blood-Retinal Barrier , Choroidal Neovascularization/metabolism , Hypoxia-Inducible Factor 1/genetics , Retina/growth & development , Retinal Neovascularization/metabolism , Vascular Endothelial Growth Factor A/genetics , Animals , Choroidal Neovascularization/etiology , Diabetic Retinopathy/metabolism , Hypoxia , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Animal , Promoter Regions, Genetic , Response Elements , Retina/metabolism , Retinal Neovascularization/etiology , Vascular Endothelial Growth Factor A/metabolism
13.
Int J Nanomedicine ; 1(3): 263-8, 2006.
Article in English | MEDLINE | ID: mdl-17717967

ABSTRACT

Age-related macular degeneration (AMD) is a major cause of severe visual loss worldwide. Neovascular (wet) AMD accounts for 90% of the visual loss associated with the disorder and vascular endothelial growth factor (VEGF) has been shown to play a major role in neovascularization and vascular permeability, the major causes of visual loss in AMD, making it an ideal target for therapeutic intervention. To utilize this strategy, pegaptanib, an aptamer that specifically binds to and blocks VEGF165, the VEGF isoform primarily responsible for abnormal vascular growth and permeability in AMD, was developed. Following encouraging preclinical trials, clinical trials showed that pegaptanib stabilized vision and reduced the risk of severe visual loss in the majority of patients with AMD, with some patients showing visual improvement. Pegaptanib has maintained a good safety profile with only occasional adverse effects. Even greater success was achieved when pegaptanib was used in combination with another therapeutic strategy, such as photodynamic therapy or bevacizumab, a pan isoform VEGF inhibitor. Further investigation of pegaptanib for the therapy of wet AMD, particularly in combination with other modes of therapy, should be encouraged.


Subject(s)
Aptamers, Nucleotide/administration & dosage , Choroidal Neovascularization/drug therapy , Clinical Trials as Topic , Macular Degeneration/drug therapy , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Humans
14.
Curr Eye Res ; 28(6): 421-6, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15512950

ABSTRACT

PURPOSE: Post-surgical macular edema is an important clinical problem resulting from breakdown of the blood-retinal barrier (BRB) after surgery. This study was designed to develop a mouse model of post-surgical BRB breakdown. METHODS: Two 25-gauge needles, one for infusion and one for aspiration, were inserted through the limbus and into the lens of one eye of adult male C57BL/6 mice. The anterior portion of the lens was aspirated and the fellow eye was untreated. At several time points after surgery, the integrity of the BRB was assessed quantitatively, using [3H]mannitol as a tracer, or qualitatively, using immunohistochemical staining for albumin. RESULTS: Eyes with partial lens extraction had a significant increase in retinal vascular leakage one day after surgery, which persisted two and three days after surgery, but by five days, was not significantly different from controls. Immunohistochemical staining for albumin demonstrated that the breech in the barrier was sufficient to allow passage of a 60kDa protein into the retina, and was localized predominantly to retinal vessels. CONCLUSIONS: Partial lens extraction in mice results in BRB breakdown (primarily the inner BRB) that is highly reproducible in the severity of leakage and its time course. This provides a valuable tool for investigation of the molecular pathogenesis and new treatment approaches for post-surgical breakdown of the BRB.


Subject(s)
Aphakia, Postcataract/complications , Blood-Retinal Barrier , Disease Models, Animal , Macular Edema/etiology , Postoperative Complications , Animals , Lens, Crystalline/surgery , Macular Edema/pathology , Male , Mice , Mice, Inbred C57BL , Retina/metabolism , Retina/pathology , Retinal Vessels/pathology , Serum Albumin/metabolism
15.
J Cell Physiol ; 195(2): 210-9, 2003 May.
Article in English | MEDLINE | ID: mdl-12652648

ABSTRACT

Breakdown of the blood-retinal barrier (BRB) occurs in several retinal diseases and is a major cause of visual loss. Vascular endothelial growth factor (VEGF) has been implicated as a cause of BRB breakdown in diabetic retinopathy and other ischemic retinopathies, and there is evidence to suggest that other vasopermeability factors may act indirectly through VEGF. In this study, we investigated the effect of several receptor kinase inhibitors on BRB breakdown resulting from VEGF, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), insulin-like growth factor-1 (IGF-1), prostaglandin E1 (PGE(1)), or PGE(2). Inhibitors of VEGF receptor kinase, including PKC412, PTK787, and SU1498, decreased VEGF-induced breakdown of the BRB. None of the inhibitors blocked leakage caused by TNF-alpha, IL-1beta, or IGF-1 and only PKC412, an inhibitor of protein kinase C (PKC) as well as VEGF and platelet-derived growth factor (PDGF) receptor kinases, decreased leakage caused by prostaglandins. Since the other inhibitors of VEGF and/or PDGF receptor kinases that do not also inhibit PKC had no effect on prostaglandin-induced breakdown of the BRB, these data implicate PKC in retinal vascular leakage caused by prostaglandins. PKC412 may be useful for treatment of post-operative and inflammatory macular edema, in which prostaglandins play a role, as well as macular edema associated with ischemic retinopathies.


Subject(s)
Blood-Retinal Barrier/drug effects , Endothelial Growth Factors/antagonists & inhibitors , Lymphokines/antagonists & inhibitors , Retina/drug effects , Retinal Diseases/drug therapy , Alprostadil/antagonists & inhibitors , Alprostadil/metabolism , Animals , Blood-Retinal Barrier/physiology , Dinoprostone/antagonists & inhibitors , Dinoprostone/metabolism , Endothelial Growth Factors/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Insulin-Like Growth Factor I/antagonists & inhibitors , Insulin-Like Growth Factor I/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-1/antagonists & inhibitors , Interleukin-1/metabolism , Lymphokines/metabolism , Male , Mice , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Retina/enzymology , Retina/physiopathology , Retinal Diseases/enzymology , Retinal Diseases/physiopathology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
16.
Brain Res Dev Brain Res ; 140(2): 169-83, 2003 Feb 16.
Article in English | MEDLINE | ID: mdl-12586423

ABSTRACT

Platelet-derived growth factor (PDGF) is necessary for the normal development of the retinal vasculature and its overexpression is likely to contribute to proliferative retinal disorders, such as proliferative vitreoretinopathy. Transgenic mice that overexpress PDGF-B in the photoreceptors (rho/PDGF-B mice) develop traction retinal detachment. In the present study, a detailed histopathological analysis was performed in rho/PDGF-B mice. In these transgenic mice, endothelial cells, pericytes, and glial cells begin to proliferate at postnatal day 7 (P7). All three cell types increase in numbers, forming a highly vascularized cell mass, which reaches a maximum thickness at P14. Cords of endothelial cells and glia invade the retina and exert traction, generating retinal folds; however, the deep capillary bed never forms. Griffonia simplicifolia isolectin B4 (GSA)-positive endothelial cells form tubes and penetrate the retina to the level of the outer plexiform layer, but they never interconnect to form the deep capillary bed. The vessels within the cell mass are patent, but have a very immature morphology. They often are thin-walled with fenestrations. Pericytes and glial cells are usually found in clusters and are not associated with the abnormal vessels. The lack of this association may account for the failure to form a mature vasculature.


Subject(s)
Photoreceptor Cells/metabolism , Proto-Oncogene Proteins c-sis/metabolism , Retina/metabolism , Retina/pathology , Retinal Detachment/etiology , Actins/metabolism , Animals , Animals, Newborn/growth & development , Animals, Newborn/metabolism , Cell Division/physiology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Glial Fibrillary Acidic Protein/metabolism , Humans , Immunohistochemistry , Mice , Mice, Transgenic/genetics , Microscopy, Electron , Muscle, Smooth/metabolism , Neuroglia/metabolism , Neuroglia/pathology , Pericytes/metabolism , Pericytes/pathology , Plant Lectins/pharmacokinetics , Proliferating Cell Nuclear Antigen/metabolism , Proto-Oncogene Proteins c-sis/genetics
17.
Curr Opin Mol Ther ; 5(6): 673-9, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14755895

ABSTRACT

Eyetech Pharmaceuticals and Pfizer are co-developing the anti-vascular endothelial growth factor aptamer, pegaptanib, as an angiogenesis inhibitor for the potential treatment of age-related macular degeneration and diabetic macular edema, in addition to other ocular diseases. Gilead was previously investigating the aptamer for the potential treatment of cancer, however, no data have been published for this indication since 1999.


Subject(s)
Angiogenesis Inhibitors , Diabetic Retinopathy/drug therapy , Macular Degeneration/drug therapy , Vascular Endothelial Growth Factor A/metabolism , Angiogenesis Inhibitors/adverse effects , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/metabolism , Angiogenesis Inhibitors/therapeutic use , Animals , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Clinical Trials, Phase III as Topic , Humans , Neovascularization, Pathologic/drug therapy , Retinal Neovascularization/drug therapy , Structure-Activity Relationship , Vascular Endothelial Growth Factor A/genetics
18.
Invest Ophthalmol Vis Sci ; 43(7): 2462-7, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12091451

ABSTRACT

PURPOSE: The purpose of this study was to develop and characterize a quantitative assay of blood-retinal barrier (BRB) function in mice and to determine the effect of several purported vasopermeability factors on the BRB. METHODS: Adult C57BL/6J mice were treated with three regimens of increasingly extensive retinal cryopexy and subsequently were given an intraperitoneal injection of 1 microCi/g body weight of [(3)H]mannitol. At several time points, the amount of radioactivity per milligram tissue was compared in retina, lung, and kidney. Time points that maximize signal-to-background differential in the retina were identified, and the ratio of counts per minute (CPM) per milligram retina to CPM per milligram lung (retina-to-lung leakage ratio, RLLR) or kidney (retina-to-renal leakage ratio, RRLR) were calculated. This technique was then used to compare the amount of BRB breakdown that occurs after intravitreous injection of vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1, prostaglandin (PG) E(1), PGE(2), interleukin (IL)-1beta, or tumor necrosis factor (TNF)-alpha. RESULTS: Twenty-four hours after retinal cryopexy, there was a higher level of radioactivity in treated than in control retinas, and the signal-to-background difference was optimal when measurements were obtained 1 hour after injection of [(3)H]mannitol. In untreated mice, the RLLR was 0.30 +/- 0.02 and the RRLR was 0.22 +/- 0.01. Twenty-four hours after one 5-second application of retinal cryopexy, the RLLR was 0.73 +/- 0.20 and the RRLR was 0.71 +/- 0.23. With increasing amounts of cryopexy, there was an increase in the RLLR and RRLR, so that after two 10-second applications, the RLLR was 1.66 +/- 0.31 and the RRLR was 1.47 +/- 0.20. Intravitreous injection of VEGF, IGF-1, PGE(1), PGE(2), IL-1beta, or TNF-alpha each caused significant increases in the RLLR and RRLR, but there were some differences in potency and time course. VEGF caused prominent BRB breakdown at 6 hours that returned to near normal by 24 hours. IL-1beta also caused relatively rapid breakdown of the BRB, but its effect was more prolonged than that caused by VEGF. There was delayed, but substantial breakdown of the BRB after injection of TNF-alpha. IGF-1, PGE(2), and PGE(1) caused less severe, relatively delayed, and more prolonged BRB breakdown. CONCLUSIONS: Measurement of the RLLR or RRLR after intraperitoneal injection of [(3)H]mannitol in mice provides a quantitative assessment of BRB function that is normalized and can therefore be compared from assay to assay. Comparison of the extent and duration of BRB breakdown after intravitreous injection of vasoactive substances shows that agents can be grouped by resultant extent and time course of leakage. Additional studies are needed to determine whether this grouping has its basis in shared mechanisms of BRB disruption.


Subject(s)
Blood-Retinal Barrier/physiology , Capillary Permeability/physiology , Retina/metabolism , Animals , Biological Transport, Active , Capillary Permeability/drug effects , Cryosurgery , Growth Substances/pharmacology , Interleukin-1/pharmacology , Kidney/physiology , Lung/physiology , Mannitol/administration & dosage , Mice , Mice, Inbred C57BL , Prostaglandins E/pharmacology , Retina/drug effects , Retina/surgery
19.
Photochem Photobiol ; 75(2): 149-58, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11883603

ABSTRACT

Laser targeted photo-occlusion (LTO) is a novel method being developed to treat choroidal neovascular membranes (CNV) in age-related and other macular degenerations. A photosensitive agent, encapsulated in heat-sensitive liposomes, is administered intravenously. A low power laser warms the targeted tissue and releases a bolus of photosensitizer. The photosensitizer is activated after it clears from the normal choriocapillaris but not from the CNV. Forty-five experimental CNV were induced in seven rats. Five weeks after LTO, complete occlusion was observed by laser targeted angiography (LTA) in 76% of treated CNV, and partial occlusion was found in the remaining 24%. The tissues outside the CNV but within the area treated by LTO showed no flow alteration and no dye leakage. All untreated CNV were patent on LTA at 5 weeks. Light microscopy and electron microscopy confirmed the results in treated and control lesions. Moreover, treated areas next to lesions showed normal photoreceptors, retinal pigment epithelium (RPE), Bruch's membrane and choriocapillaris. These results indicate that LTO may improve current photodynamic therapy by alleviating the need for repeated treatments and by avoiding the long-term risks associated with damage to the RPE and occlusion of normal choriocapillaries.


Subject(s)
Choroidal Neovascularization/drug therapy , Laser Coagulation/methods , Animals , Choroidal Neovascularization/complications , Choroidal Neovascularization/therapy , Disease Models, Animal , Laser Coagulation/adverse effects , Laser Coagulation/standards , Laser Therapy , Lasers/standards , Photochemotherapy/adverse effects , Photochemotherapy/methods , Photochemotherapy/standards , Photosensitizing Agents/administration & dosage , Photosensitizing Agents/therapeutic use , Rats , Rats, Inbred Strains , Treatment Outcome
SELECTION OF CITATIONS
SEARCH DETAIL
...