Limits...
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.

Show MeSH

Related in: MedlinePlus

K5 expression from K5-NP in vitro. ARPE19 cells were grown to 70% confluence in a medium containing 5% FBS. The culture medium was replaced by a serum-free medium. Control-NP and K5-NP were separately added into the medium to 1 μg/ml (plasmid DNA concentration) and incubated with the cells for 72 h. A: The medium was collected and concentrated. Total protein concentrations in the media were measured using the Bradford assay. The same amount of total proteins (20 μg) was applied for Western blot analysis separately using an antibody specific for human K5 and an anti–His-tag antibody. Lane 1, molecular weight markers; lane 2, medium from cells treated with K5-NP; lane 3, untreated cells; lane 4, treated with control-NP; lane 5, purified K5 peptide as positive control. B and C: ARPE19 cells were grown overnight on glass slides, treated with K5-NP and control-NP at 1 μg/ml for 72 h, washed, and fixed. The cells were immunostained with a monoclonal antibody against the His-tag using a 3,3′-diaminobenzidine color reaction that shows brown color in cells with positive immunostaining. B: 100×, C: 400×. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2712783&req=5

Figure 1: K5 expression from K5-NP in vitro. ARPE19 cells were grown to 70% confluence in a medium containing 5% FBS. The culture medium was replaced by a serum-free medium. Control-NP and K5-NP were separately added into the medium to 1 μg/ml (plasmid DNA concentration) and incubated with the cells for 72 h. A: The medium was collected and concentrated. Total protein concentrations in the media were measured using the Bradford assay. The same amount of total proteins (20 μg) was applied for Western blot analysis separately using an antibody specific for human K5 and an anti–His-tag antibody. Lane 1, molecular weight markers; lane 2, medium from cells treated with K5-NP; lane 3, untreated cells; lane 4, treated with control-NP; lane 5, purified K5 peptide as positive control. B and C: ARPE19 cells were grown overnight on glass slides, treated with K5-NP and control-NP at 1 μg/ml for 72 h, washed, and fixed. The cells were immunostained with a monoclonal antibody against the His-tag using a 3,3′-diaminobenzidine color reaction that shows brown color in cells with positive immunostaining. B: 100×, C: 400×. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: ARPE19 cells were transfected with 10 μg of K5-NP or Control-NP for 3 days. The concentrated conditioned serum-free medium from the transfected cells was blotted separately with the anti-K5 and anti–His-tag antibodies. Both of the antibodies detected significant amounts of K5 with the expected molecular weight secreted from the cells transfected with K5-NP, but not from the cells transfected with Control-NP (Fig. 1A).


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 from K5-NP in vitro. ARPE19 cells were grown to 70% confluence in a medium containing 5% FBS. The culture medium was replaced by a serum-free medium. Control-NP and K5-NP were separately added into the medium to 1 μg/ml (plasmid DNA concentration) and incubated with the cells for 72 h. A: The medium was collected and concentrated. Total protein concentrations in the media were measured using the Bradford assay. The same amount of total proteins (20 μg) was applied for Western blot analysis separately using an antibody specific for human K5 and an anti–His-tag antibody. Lane 1, molecular weight markers; lane 2, medium from cells treated with K5-NP; lane 3, untreated cells; lane 4, treated with control-NP; lane 5, purified K5 peptide as positive control. B and C: ARPE19 cells were grown overnight on glass slides, treated with K5-NP and control-NP at 1 μg/ml for 72 h, washed, and fixed. The cells were immunostained with a monoclonal antibody against the His-tag using a 3,3′-diaminobenzidine color reaction that shows brown color in cells with positive immunostaining. B: 100×, C: 400×. (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
getmorefigures.php?uid=PMC2712783&req=5

Figure 1: K5 expression from K5-NP in vitro. ARPE19 cells were grown to 70% confluence in a medium containing 5% FBS. The culture medium was replaced by a serum-free medium. Control-NP and K5-NP were separately added into the medium to 1 μg/ml (plasmid DNA concentration) and incubated with the cells for 72 h. A: The medium was collected and concentrated. Total protein concentrations in the media were measured using the Bradford assay. The same amount of total proteins (20 μg) was applied for Western blot analysis separately using an antibody specific for human K5 and an anti–His-tag antibody. Lane 1, molecular weight markers; lane 2, medium from cells treated with K5-NP; lane 3, untreated cells; lane 4, treated with control-NP; lane 5, purified K5 peptide as positive control. B and C: ARPE19 cells were grown overnight on glass slides, treated with K5-NP and control-NP at 1 μg/ml for 72 h, washed, and fixed. The cells were immunostained with a monoclonal antibody against the His-tag using a 3,3′-diaminobenzidine color reaction that shows brown color in cells with positive immunostaining. B: 100×, C: 400×. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: ARPE19 cells were transfected with 10 μg of K5-NP or Control-NP for 3 days. The concentrated conditioned serum-free medium from the transfected cells was blotted separately with the anti-K5 and anti–His-tag antibodies. Both of the antibodies detected significant amounts of K5 with the expected molecular weight secreted from the cells transfected with K5-NP, but not from the cells transfected with Control-NP (Fig. 1A).

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