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Nanoparticle-mediated expression of an angiogenic inhibitor ameliorates ischemia-induced retinal neovascularization and diabetes-induced retinal vascular leakage.

Park K, Chen Y, Hu Y, Mayo AS, Kompella UB, Longeras R, Ma JX - Diabetes (2009)

Bottom Line: Injection of K5-NP significantly reduced retinal vascular leakage and attenuated retinal neovascularization, when compared with the contralateral eyes injected with Control-NP in oxygen-induced retinopathy rats.No toxicities of K5-NP were detected to retinal structure and function.K5-NP mediates efficient and sustained K5 expression in the retina and has therapeutic potential for diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

ABSTRACT

Objective: The aim of the study is to evaluate the effect of nanoparticle-mediated gene delivery of angiogenic inhibitors on retinal inflammation, vascular leakage, and neovascularization in diabetic retinopathy.

Research design and methods: An expression plasmid of plasminogen kringle 5 (K5), a natural angiogenic inhibitor, was encapsulated with poly(lactide-coglycolide) to form K5 nanoparticles (K5-NP). Expression of K5 was determined by Western blot analysis and immunohistochemistry, and retinal vascular leakage was measured by permeability assay. Retinal neovascularization was evaluated using fluorescein-angiography and counting preretinal vascular cells in rats with oxygen-induced retinopathy. Effects of K5-NP on retinal inflammation were evaluated in streptozotocin-induced diabetic rats by leukostasis assay and Western blot analysis of intracellular adhesion molecule and vascular endothelial growth factor. Possible toxicities of K5-NP were evaluated using histology examination, retinal thickness measurement, and electroretinogram recording.

Results: K5-NP mediated efficient expression of K5 and specifically inhibited growth of endothelial cells. An intravitreal injection of K5-NP resulted in high-level expression of K5 in the inner retina of rats during the 4 weeks they were analyzed. Injection of K5-NP significantly reduced retinal vascular leakage and attenuated retinal neovascularization, when compared with the contralateral eyes injected with Control-NP in oxygen-induced retinopathy rats. K5-NP attenuated vascular endothelial growth factor and intracellular adhesion molecule-1 overexpression and reduced leukostasis and vascular leakage for at least 4 weeks after a single injection in the retina of streptozotocin-induced diabetic rats. No toxicities of K5-NP were detected to retinal structure and function.

Conclusions: K5-NP mediates efficient and sustained K5 expression in the retina and has therapeutic potential for diabetic retinopathy.

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Related in: MedlinePlus

K5 expression in the rat retina after an intravitreal injection of K5-NP. A–D: K5-NP was injected into the vitreous of the right eyes and control-NP into the contralateral eyes of four OIR rats at P12. K5 expression was examined at P18 by immunohistochemistry in ocular sections using an anti–His-tag antibody (green). The nuclei were counterstained with DAPI (red). A and C: Representative immunostaining images from the eyes injected with K5-NP (A) and control-NP (C). B and D: Phase contrast images of the same areas shown in A and C, respectively. Scale bar: 20 μm. E: K5-NP and control-NP were separately injected into the vitreous. The retinas were dissected at 1, 2, 3, and 4 weeks after the injection of K5-NP or control-NP (three rats per time point). The same amount of proteins (100 μg) from each retina was loaded for Western blot analysis using the anti–His-tag antibody. The same membrane was striped and re-blotted with an anti–β-actin antibody. No K5 expression was found in the retinas injected with control-NP, whereas K5 expression was detected in all of the retinas with the K5-NP injection. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: K5 expression in the rat retina after an intravitreal injection of K5-NP. A–D: K5-NP was injected into the vitreous of the right eyes and control-NP into the contralateral eyes of four OIR rats at P12. K5 expression was examined at P18 by immunohistochemistry in ocular sections using an anti–His-tag antibody (green). The nuclei were counterstained with DAPI (red). A and C: Representative immunostaining images from the eyes injected with K5-NP (A) and control-NP (C). B and D: Phase contrast images of the same areas shown in A and C, respectively. Scale bar: 20 μm. E: K5-NP and control-NP were separately injected into the vitreous. The retinas were dissected at 1, 2, 3, and 4 weeks after the injection of K5-NP or control-NP (three rats per time point). The same amount of proteins (100 μg) from each retina was loaded for Western blot analysis using the anti–His-tag antibody. The same membrane was striped and re-blotted with an anti–β-actin antibody. No K5 expression was found in the retinas injected with control-NP, whereas K5 expression was detected in all of the retinas with the K5-NP injection. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: To examine the expression of K5 from K5-NP in the retina, K5-NP was injected into the vitreous of the right eye (8.8 μg/eye) and the same amount of Control-NP into the left vitreous of four OIR rats at age of postnatal day 12 (P12) after exposure to 75% oxygen. At age P18, the immunohistochemistry was performed on retinal sections using the anti–His-tag antibody. The immunostaining detected high levels of K5 expression in the inner retina in the eyes injected with K5-NP, but not in the contralateral control eyes (Fig. 3A–D).


Nanoparticle-mediated expression of an angiogenic inhibitor ameliorates ischemia-induced retinal neovascularization and diabetes-induced retinal vascular leakage.

Park K, Chen Y, Hu Y, Mayo AS, Kompella UB, Longeras R, Ma JX - Diabetes (2009)

K5 expression in the rat retina after an intravitreal injection of K5-NP. A–D: K5-NP was injected into the vitreous of the right eyes and control-NP into the contralateral eyes of four OIR rats at P12. K5 expression was examined at P18 by immunohistochemistry in ocular sections using an anti–His-tag antibody (green). The nuclei were counterstained with DAPI (red). A and C: Representative immunostaining images from the eyes injected with K5-NP (A) and control-NP (C). B and D: Phase contrast images of the same areas shown in A and C, respectively. Scale bar: 20 μm. E: K5-NP and control-NP were separately injected into the vitreous. The retinas were dissected at 1, 2, 3, and 4 weeks after the injection of K5-NP or control-NP (three rats per time point). The same amount of proteins (100 μg) from each retina was loaded for Western blot analysis using the anti–His-tag antibody. The same membrane was striped and re-blotted with an anti–β-actin antibody. No K5 expression was found in the retinas injected with control-NP, whereas K5 expression was detected in all of the retinas with the K5-NP injection. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: K5 expression in the rat retina after an intravitreal injection of K5-NP. A–D: K5-NP was injected into the vitreous of the right eyes and control-NP into the contralateral eyes of four OIR rats at P12. K5 expression was examined at P18 by immunohistochemistry in ocular sections using an anti–His-tag antibody (green). The nuclei were counterstained with DAPI (red). A and C: Representative immunostaining images from the eyes injected with K5-NP (A) and control-NP (C). B and D: Phase contrast images of the same areas shown in A and C, respectively. Scale bar: 20 μm. E: K5-NP and control-NP were separately injected into the vitreous. The retinas were dissected at 1, 2, 3, and 4 weeks after the injection of K5-NP or control-NP (three rats per time point). The same amount of proteins (100 μg) from each retina was loaded for Western blot analysis using the anti–His-tag antibody. The same membrane was striped and re-blotted with an anti–β-actin antibody. No K5 expression was found in the retinas injected with control-NP, whereas K5 expression was detected in all of the retinas with the K5-NP injection. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: To examine the expression of K5 from K5-NP in the retina, K5-NP was injected into the vitreous of the right eye (8.8 μg/eye) and the same amount of Control-NP into the left vitreous of four OIR rats at age of postnatal day 12 (P12) after exposure to 75% oxygen. At age P18, the immunohistochemistry was performed on retinal sections using the anti–His-tag antibody. The immunostaining detected high levels of K5 expression in the inner retina in the eyes injected with K5-NP, but not in the contralateral control eyes (Fig. 3A–D).

Bottom Line: Injection of K5-NP significantly reduced retinal vascular leakage and attenuated retinal neovascularization, when compared with the contralateral eyes injected with Control-NP in oxygen-induced retinopathy rats.No toxicities of K5-NP were detected to retinal structure and function.K5-NP mediates efficient and sustained K5 expression in the retina and has therapeutic potential for diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

ABSTRACT

Objective: The aim of the study is to evaluate the effect of nanoparticle-mediated gene delivery of angiogenic inhibitors on retinal inflammation, vascular leakage, and neovascularization in diabetic retinopathy.

Research design and methods: An expression plasmid of plasminogen kringle 5 (K5), a natural angiogenic inhibitor, was encapsulated with poly(lactide-coglycolide) to form K5 nanoparticles (K5-NP). Expression of K5 was determined by Western blot analysis and immunohistochemistry, and retinal vascular leakage was measured by permeability assay. Retinal neovascularization was evaluated using fluorescein-angiography and counting preretinal vascular cells in rats with oxygen-induced retinopathy. Effects of K5-NP on retinal inflammation were evaluated in streptozotocin-induced diabetic rats by leukostasis assay and Western blot analysis of intracellular adhesion molecule and vascular endothelial growth factor. Possible toxicities of K5-NP were evaluated using histology examination, retinal thickness measurement, and electroretinogram recording.

Results: K5-NP mediated efficient expression of K5 and specifically inhibited growth of endothelial cells. An intravitreal injection of K5-NP resulted in high-level expression of K5 in the inner retina of rats during the 4 weeks they were analyzed. Injection of K5-NP significantly reduced retinal vascular leakage and attenuated retinal neovascularization, when compared with the contralateral eyes injected with Control-NP in oxygen-induced retinopathy rats. K5-NP attenuated vascular endothelial growth factor and intracellular adhesion molecule-1 overexpression and reduced leukostasis and vascular leakage for at least 4 weeks after a single injection in the retina of streptozotocin-induced diabetic rats. No toxicities of K5-NP were detected to retinal structure and function.

Conclusions: K5-NP mediates efficient and sustained K5 expression in the retina and has therapeutic potential for diabetic retinopathy.

Show MeSH
Related in: MedlinePlus