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Targeted decorin gene therapy delivered with adeno-associated virus effectively retards corneal neovascularization in vivo.

Mohan RR, Tovey JC, Sharma A, Schultz GS, Cowden JW, Tandon A - PLoS ONE (2011)

Bottom Line: An established rabbit CNV model was used.Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations.Selective decorin delivery into stroma showed significant 52% (p<0.05), 66% (p<0.001), and 63% (p<0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis.

View Article: PubMed Central - PubMed

Affiliation: Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States of America. mohanr@health.missouri.edu

ABSTRACT
Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 µl; 5×10(12) vg/ml) application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p<0.05), 66% (p<0.001), and 63% (p<0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57-65, p<0.5), and CD31 immunoblotting (62-67%, p<0.05) supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic) and up-regulated PEDF (anti-angiogenic) genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5-mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients.

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Representative H&E staining images showing anti-angiogenic efficiency of decorin gene therapy in rabbit corneas.The decorin-delivered rabbit corneas (B and D) showed a statistically significant (p<0.01) reduction in vasculature area, density, and blood vessels number, length and diameter compared to control (no decorin-delivered) rabbit corneas (A and C) collected 14 days after VEGF implantation. The images shown in panels A and B are from the corneal sections obtained from the peripheral region of the cornea closer to the limbus whereas the images shown in panels C and D are from the corneal sections proximal to the VEGF pellet. Scale bar denotes 100 µm. dcn = decorin.
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pone-0026432-g004: Representative H&E staining images showing anti-angiogenic efficiency of decorin gene therapy in rabbit corneas.The decorin-delivered rabbit corneas (B and D) showed a statistically significant (p<0.01) reduction in vasculature area, density, and blood vessels number, length and diameter compared to control (no decorin-delivered) rabbit corneas (A and C) collected 14 days after VEGF implantation. The images shown in panels A and B are from the corneal sections obtained from the peripheral region of the cornea closer to the limbus whereas the images shown in panels C and D are from the corneal sections proximal to the VEGF pellet. Scale bar denotes 100 µm. dcn = decorin.

Mentions: Figure 4 shows localization and density of blood vessels in control and decorin-delivered tissue sections of rabbit corneas collected 14 days after VEGF-pellet implantation. Serial sections were prepared. As expected, corneal sections obtained from the peripheral region of the cornea closer to the limbus showed many large-diameter blood vessels (Fig 4A and 4B) whereas corneal sections prepared from the region closer to VEGF pellet showed numerous small-diameter blood vessels (Fig 4C and 4D). The H&E staining performed in the corneal sections of the AAV5-naked and AAV5-dcn vector groups obtained from the same peripheral regions showed markedly less number and reduced-diameter blood vessels in the decorin-delivered rabbit corneas (Fig 4B) compared to the control cornea (Fig. 4A). Similar anti-angiogenic effects of decorin gene therapy were noted in the H&E stained corneal sections prepared from the region closer to the VEGF pellet as decorin-delivered rabbit corneas (Fig 4D) demonstrated significantly (p<0.01) fewer and thin-diameter blood vessels compared to control corneas (Fig. 4C).


Targeted decorin gene therapy delivered with adeno-associated virus effectively retards corneal neovascularization in vivo.

Mohan RR, Tovey JC, Sharma A, Schultz GS, Cowden JW, Tandon A - PLoS ONE (2011)

Representative H&E staining images showing anti-angiogenic efficiency of decorin gene therapy in rabbit corneas.The decorin-delivered rabbit corneas (B and D) showed a statistically significant (p<0.01) reduction in vasculature area, density, and blood vessels number, length and diameter compared to control (no decorin-delivered) rabbit corneas (A and C) collected 14 days after VEGF implantation. The images shown in panels A and B are from the corneal sections obtained from the peripheral region of the cornea closer to the limbus whereas the images shown in panels C and D are from the corneal sections proximal to the VEGF pellet. Scale bar denotes 100 µm. dcn = decorin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198476&req=5

pone-0026432-g004: Representative H&E staining images showing anti-angiogenic efficiency of decorin gene therapy in rabbit corneas.The decorin-delivered rabbit corneas (B and D) showed a statistically significant (p<0.01) reduction in vasculature area, density, and blood vessels number, length and diameter compared to control (no decorin-delivered) rabbit corneas (A and C) collected 14 days after VEGF implantation. The images shown in panels A and B are from the corneal sections obtained from the peripheral region of the cornea closer to the limbus whereas the images shown in panels C and D are from the corneal sections proximal to the VEGF pellet. Scale bar denotes 100 µm. dcn = decorin.
Mentions: Figure 4 shows localization and density of blood vessels in control and decorin-delivered tissue sections of rabbit corneas collected 14 days after VEGF-pellet implantation. Serial sections were prepared. As expected, corneal sections obtained from the peripheral region of the cornea closer to the limbus showed many large-diameter blood vessels (Fig 4A and 4B) whereas corneal sections prepared from the region closer to VEGF pellet showed numerous small-diameter blood vessels (Fig 4C and 4D). The H&E staining performed in the corneal sections of the AAV5-naked and AAV5-dcn vector groups obtained from the same peripheral regions showed markedly less number and reduced-diameter blood vessels in the decorin-delivered rabbit corneas (Fig 4B) compared to the control cornea (Fig. 4A). Similar anti-angiogenic effects of decorin gene therapy were noted in the H&E stained corneal sections prepared from the region closer to the VEGF pellet as decorin-delivered rabbit corneas (Fig 4D) demonstrated significantly (p<0.01) fewer and thin-diameter blood vessels compared to control corneas (Fig. 4C).

Bottom Line: An established rabbit CNV model was used.Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations.Selective decorin delivery into stroma showed significant 52% (p<0.05), 66% (p<0.001), and 63% (p<0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis.

View Article: PubMed Central - PubMed

Affiliation: Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States of America. mohanr@health.missouri.edu

ABSTRACT
Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 µl; 5×10(12) vg/ml) application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p<0.05), 66% (p<0.001), and 63% (p<0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57-65, p<0.5), and CD31 immunoblotting (62-67%, p<0.05) supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic) and up-regulated PEDF (anti-angiogenic) genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5-mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients.

Show MeSH
Related in: MedlinePlus