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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

Blockade of nuclear translocation of HIF-1α by K5-NP. A: BRCECs were treated with K5-NP or control-NP for 48 h under normoxia or hypoxia (1% oxygen). The cytosol and nucleus were isolated, and the same amount of proteins (30 μg) from each sample was used for Western blot analysis using an anti–HIF-1α antibody. The membranes were stripped and re-blotted with antibodies for β-actin and nuclear protein TBP. Cytosolic and nuclear levels of HIF-1α were semi-quantified by densitometry, normalized by β-actin and TBP, respectively, and expressed as percent of nondiabetic control (mean ± SD, n = 3). B–G: Three OIR rats received an intravitreal injection of control-NP in the left eye (B–D) and K5-NP into the right eye (E–G) at P12. The eyes were fixed and sectioned at P16. The retinal sections were stained with the antibody specific for HIF-1α (green) and the nuclei counterstained with DAPI (red). B and E: HIF-1α immunostaining; C and F: DAPI staining of the nuclei; D and G: merged images of HIF-1α and DAPI staining. Note that the nuclei with increased HIF-1α signal superimposed on DAPI staining show orange color in D. White arrows indicate different intensities of HIF-1α staining in the nuclei of inner retinal cells in D and G. Scale bar: 10 μm. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 9: Blockade of nuclear translocation of HIF-1α by K5-NP. A: BRCECs were treated with K5-NP or control-NP for 48 h under normoxia or hypoxia (1% oxygen). The cytosol and nucleus were isolated, and the same amount of proteins (30 μg) from each sample was used for Western blot analysis using an anti–HIF-1α antibody. The membranes were stripped and re-blotted with antibodies for β-actin and nuclear protein TBP. Cytosolic and nuclear levels of HIF-1α were semi-quantified by densitometry, normalized by β-actin and TBP, respectively, and expressed as percent of nondiabetic control (mean ± SD, n = 3). B–G: Three OIR rats received an intravitreal injection of control-NP in the left eye (B–D) and K5-NP into the right eye (E–G) at P12. The eyes were fixed and sectioned at P16. The retinal sections were stained with the antibody specific for HIF-1α (green) and the nuclei counterstained with DAPI (red). B and E: HIF-1α immunostaining; C and F: DAPI staining of the nuclei; D and G: merged images of HIF-1α and DAPI staining. Note that the nuclei with increased HIF-1α signal superimposed on DAPI staining show orange color in D. White arrows indicate different intensities of HIF-1α staining in the nuclei of inner retinal cells in D and G. Scale bar: 10 μm. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: To investigate the mechanism responsible for the anti-angiogenic activity of K5-NP, we have measured the effect of K5-NP on HIF-1 activation, since HIF-1 is the key transcription factor activating VEGF and is known to play a key role in retinal neovascularization (7). HIF-1α nuclear translocation is a critical step in the activation of HIF-1, and thus, we measured cytosolic and nuclear levels of HIF-1α in the human Müller cell line treated with K5-NP under hypoxia. As shown by Western blot analysis of isolated nuclear proteins, exposure of the cells to hypoxia (1% oxygen) increased HIF-1α levels in the nuclei (Fig. 9A). K5-NP, but not Control-NP, completely blocked the increase of nuclear HIF-1α levels under hypoxia (Fig. 9A).


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)

Blockade of nuclear translocation of HIF-1α by K5-NP. A: BRCECs were treated with K5-NP or control-NP for 48 h under normoxia or hypoxia (1% oxygen). The cytosol and nucleus were isolated, and the same amount of proteins (30 μg) from each sample was used for Western blot analysis using an anti–HIF-1α antibody. The membranes were stripped and re-blotted with antibodies for β-actin and nuclear protein TBP. Cytosolic and nuclear levels of HIF-1α were semi-quantified by densitometry, normalized by β-actin and TBP, respectively, and expressed as percent of nondiabetic control (mean ± SD, n = 3). B–G: Three OIR rats received an intravitreal injection of control-NP in the left eye (B–D) and K5-NP into the right eye (E–G) at P12. The eyes were fixed and sectioned at P16. The retinal sections were stained with the antibody specific for HIF-1α (green) and the nuclei counterstained with DAPI (red). B and E: HIF-1α immunostaining; C and F: DAPI staining of the nuclei; D and G: merged images of HIF-1α and DAPI staining. Note that the nuclei with increased HIF-1α signal superimposed on DAPI staining show orange color in D. White arrows indicate different intensities of HIF-1α staining in the nuclei of inner retinal cells in D and G. Scale bar: 10 μm. (A high-quality digital representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Figure 9: Blockade of nuclear translocation of HIF-1α by K5-NP. A: BRCECs were treated with K5-NP or control-NP for 48 h under normoxia or hypoxia (1% oxygen). The cytosol and nucleus were isolated, and the same amount of proteins (30 μg) from each sample was used for Western blot analysis using an anti–HIF-1α antibody. The membranes were stripped and re-blotted with antibodies for β-actin and nuclear protein TBP. Cytosolic and nuclear levels of HIF-1α were semi-quantified by densitometry, normalized by β-actin and TBP, respectively, and expressed as percent of nondiabetic control (mean ± SD, n = 3). B–G: Three OIR rats received an intravitreal injection of control-NP in the left eye (B–D) and K5-NP into the right eye (E–G) at P12. The eyes were fixed and sectioned at P16. The retinal sections were stained with the antibody specific for HIF-1α (green) and the nuclei counterstained with DAPI (red). B and E: HIF-1α immunostaining; C and F: DAPI staining of the nuclei; D and G: merged images of HIF-1α and DAPI staining. Note that the nuclei with increased HIF-1α signal superimposed on DAPI staining show orange color in D. White arrows indicate different intensities of HIF-1α staining in the nuclei of inner retinal cells in D and G. Scale bar: 10 μm. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: To investigate the mechanism responsible for the anti-angiogenic activity of K5-NP, we have measured the effect of K5-NP on HIF-1 activation, since HIF-1 is the key transcription factor activating VEGF and is known to play a key role in retinal neovascularization (7). HIF-1α nuclear translocation is a critical step in the activation of HIF-1, and thus, we measured cytosolic and nuclear levels of HIF-1α in the human Müller cell line treated with K5-NP under hypoxia. As shown by Western blot analysis of isolated nuclear proteins, exposure of the cells to hypoxia (1% oxygen) increased HIF-1α levels in the nuclei (Fig. 9A). K5-NP, but not Control-NP, completely blocked the increase of nuclear HIF-1α levels under hypoxia (Fig. 9A).

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