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Silencing of Rac1 expression via RNA interference inhibits retinal neovascularization in rats.

Li J, Li Y, Zhang M, Hu Z - Mol. Vis. (2012)

Bottom Line: Two weeks after transfection, the neonatal vessels were tested using fluorescein isothiocyanate-dextran retinal angiography.The number of endothelial cells beyond the internal limiting membrane was counted with hematoxylin and eosin staining.In the shRNA interference group, the mean number of endothelial cells beyond the internal limiting membrane was significantly higher than that in the positive control group or the interference negative control group (p<0.05).

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Second People’s Hospital of Yunnan Province, Kunming, China. juanjuanlicn@yeah.net

ABSTRACT

Purpose: To investigate the inhibitory effect of Ras-related C3 botulinum toxin substrate 1-small interfering RNA (Rac1-siRNA) on retinal neovascularization in a rat model.

Methods: Rac1-short hairpin RNA (shRNA) was synthesized, constructed, and transfected into HeLa cells. Reverse transcription polymerase chain reaction was then conducted to test for Rac1 gene expression. Retinal vein obstruction was performed in 25 Sprague-Dawley rats using the retinal photodynamic method. The vitrea bulbus of the eye in the shRNA rats group was transfected with the Rac1-shRNA vector, the other eye in the blank control group was transfected with the blank vector, and the interference control group was prepared by transfecting the Rac1-shRNA vector. Two weeks after transfection, the neonatal vessels were tested using fluorescein isothiocyanate-dextran retinal angiography. The number of endothelial cells beyond the internal limiting membrane was counted with hematoxylin and eosin staining.

Results: A large area of neovascularization and fluorescein isothiocyanate leakage was found in the positive control group. However, a small area of neovascularization and a little fluorescence leakage were found in the shRNA group, whereas the retinal vessels were normal in the negative control interference group. In the shRNA interference group, the mean number of endothelial cells beyond the internal limiting membrane was significantly higher than that in the positive control group or the interference negative control group (p<0.05).

Conclusions: Silencing Rac1 expression with RNA interference inhibits retinal neovascularization in rats.

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Related in: MedlinePlus

The nucleus number beyond retinal inner limiting membrane after HE staining. A: The image of the retina in the negative control group under a microscope. The endothelial cells were neatly arranged below the inner limiting membrane. B: The image of the retina in the positive control group under microscope. The endothelial cells broke through the inner limiting membrane and grew in a disorganized manner. C: The image of the retina in the short hairpin RNA group under a microscope. The number of endothelial cells increased, but only a minority of them broke through the inner limiting membrane.
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f3: The nucleus number beyond retinal inner limiting membrane after HE staining. A: The image of the retina in the negative control group under a microscope. The endothelial cells were neatly arranged below the inner limiting membrane. B: The image of the retina in the positive control group under microscope. The endothelial cells broke through the inner limiting membrane and grew in a disorganized manner. C: The image of the retina in the short hairpin RNA group under a microscope. The number of endothelial cells increased, but only a minority of them broke through the inner limiting membrane.

Mentions: The structures of the retinal sections from the three groups were clearly observed. Few vascular endothelial cells beyond the inner limiting membrane (Figure 3A) were observed, with a mean of 1.08±0.26 in the negative control group. The number of cells beyond the inner limiting membrane in the positive control group was higher, with a mean of 20.42 ± 2.36. However, the cells were arranged in a disorganized manner (Figure 3B). The number of vascular endothelial cells increased, but only a few cells beyond the inner limiting membrane were found, with a mean of 11.14±1.10 (Figure 3C). The differences among groups were statistically significant (F=47.168, p=0.000), and the differences in the number of cells beyond the inner limiting membrane between groups were statistically significant (F=23.768, p=0.002).


Silencing of Rac1 expression via RNA interference inhibits retinal neovascularization in rats.

Li J, Li Y, Zhang M, Hu Z - Mol. Vis. (2012)

The nucleus number beyond retinal inner limiting membrane after HE staining. A: The image of the retina in the negative control group under a microscope. The endothelial cells were neatly arranged below the inner limiting membrane. B: The image of the retina in the positive control group under microscope. The endothelial cells broke through the inner limiting membrane and grew in a disorganized manner. C: The image of the retina in the short hairpin RNA group under a microscope. The number of endothelial cells increased, but only a minority of them broke through the inner limiting membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369893&req=5

f3: The nucleus number beyond retinal inner limiting membrane after HE staining. A: The image of the retina in the negative control group under a microscope. The endothelial cells were neatly arranged below the inner limiting membrane. B: The image of the retina in the positive control group under microscope. The endothelial cells broke through the inner limiting membrane and grew in a disorganized manner. C: The image of the retina in the short hairpin RNA group under a microscope. The number of endothelial cells increased, but only a minority of them broke through the inner limiting membrane.
Mentions: The structures of the retinal sections from the three groups were clearly observed. Few vascular endothelial cells beyond the inner limiting membrane (Figure 3A) were observed, with a mean of 1.08±0.26 in the negative control group. The number of cells beyond the inner limiting membrane in the positive control group was higher, with a mean of 20.42 ± 2.36. However, the cells were arranged in a disorganized manner (Figure 3B). The number of vascular endothelial cells increased, but only a few cells beyond the inner limiting membrane were found, with a mean of 11.14±1.10 (Figure 3C). The differences among groups were statistically significant (F=47.168, p=0.000), and the differences in the number of cells beyond the inner limiting membrane between groups were statistically significant (F=23.768, p=0.002).

Bottom Line: Two weeks after transfection, the neonatal vessels were tested using fluorescein isothiocyanate-dextran retinal angiography.The number of endothelial cells beyond the internal limiting membrane was counted with hematoxylin and eosin staining.In the shRNA interference group, the mean number of endothelial cells beyond the internal limiting membrane was significantly higher than that in the positive control group or the interference negative control group (p<0.05).

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Second People’s Hospital of Yunnan Province, Kunming, China. juanjuanlicn@yeah.net

ABSTRACT

Purpose: To investigate the inhibitory effect of Ras-related C3 botulinum toxin substrate 1-small interfering RNA (Rac1-siRNA) on retinal neovascularization in a rat model.

Methods: Rac1-short hairpin RNA (shRNA) was synthesized, constructed, and transfected into HeLa cells. Reverse transcription polymerase chain reaction was then conducted to test for Rac1 gene expression. Retinal vein obstruction was performed in 25 Sprague-Dawley rats using the retinal photodynamic method. The vitrea bulbus of the eye in the shRNA rats group was transfected with the Rac1-shRNA vector, the other eye in the blank control group was transfected with the blank vector, and the interference control group was prepared by transfecting the Rac1-shRNA vector. Two weeks after transfection, the neonatal vessels were tested using fluorescein isothiocyanate-dextran retinal angiography. The number of endothelial cells beyond the internal limiting membrane was counted with hematoxylin and eosin staining.

Results: A large area of neovascularization and fluorescein isothiocyanate leakage was found in the positive control group. However, a small area of neovascularization and a little fluorescence leakage were found in the shRNA group, whereas the retinal vessels were normal in the negative control interference group. In the shRNA interference group, the mean number of endothelial cells beyond the internal limiting membrane was significantly higher than that in the positive control group or the interference negative control group (p<0.05).

Conclusions: Silencing Rac1 expression with RNA interference inhibits retinal neovascularization in rats.

Show MeSH
Related in: MedlinePlus