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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 optical coherence tomography (A and B) and transmission electron microscopy (C and D) images of rabbits receiving AAV5-gfp or AAV5-dcn.Optical coherence tomography was performed in live rabbits on day 10 after the AAV5-gfp or AAV5-dcn application and transmission electron microscopy was performed in corneas collected 14 days after AAV5-gfp or AAV5-dcn application. The optical scans of no decorin-delivered (A) and decorin-delivered (B) cornea suggest that tissue-selective targeted delivery of decorin gene with AAV5 in the rabbit stroma does not induce changes in corneal structure, thickness or shape. The TEM did not detect significant differences in collagen fibril diameter and arrangement in the naive (C) and decorin-delivered rabbit corneas (D) indicating that localized therapeutic levels of decorin delivery in the cornea does not appear compromise corneal health. dcn = decorin.
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pone-0026432-g008: Representative optical coherence tomography (A and B) and transmission electron microscopy (C and D) images of rabbits receiving AAV5-gfp or AAV5-dcn.Optical coherence tomography was performed in live rabbits on day 10 after the AAV5-gfp or AAV5-dcn application and transmission electron microscopy was performed in corneas collected 14 days after AAV5-gfp or AAV5-dcn application. The optical scans of no decorin-delivered (A) and decorin-delivered (B) cornea suggest that tissue-selective targeted delivery of decorin gene with AAV5 in the rabbit stroma does not induce changes in corneal structure, thickness or shape. The TEM did not detect significant differences in collagen fibril diameter and arrangement in the naive (C) and decorin-delivered rabbit corneas (D) indicating that localized therapeutic levels of decorin delivery in the cornea does not appear compromise corneal health. dcn = decorin.

Mentions: Visual and slit-lamp clinical examination performed in the eyes of live rabbits did not detect any abnormality in non decorin-delivered or decorin-delivered corneas with AAV5 at all tested time points. No haziness, swelling, redness or infection was detected in visual clinical eye examination except in the region where VEGF pellet was implanted. As expected, several blood vessels sprouting from the limbus towards the VEGF pellet were observed in clinical examination. Optical coherence tomography did not detect alteration in corneal structure, thickness or shape of the stroma in decorin-delivered and no decorin-delivered rabbit corneas (Fig 8A and 8B). Furthermore, TEM analysis of naive and decorin-delivered rabbit corneas did not detect any significant differences between the collagen fibril diameter and arrangement in the stroma (Fig 8C and 8D). Slit-lamp, optical coherence tomography and TEM data allowed us to infer that targeted decorin gene transfer into stroma does not affect optical and structural properties of the cornea, and consequently visual function.


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 optical coherence tomography (A and B) and transmission electron microscopy (C and D) images of rabbits receiving AAV5-gfp or AAV5-dcn.Optical coherence tomography was performed in live rabbits on day 10 after the AAV5-gfp or AAV5-dcn application and transmission electron microscopy was performed in corneas collected 14 days after AAV5-gfp or AAV5-dcn application. The optical scans of no decorin-delivered (A) and decorin-delivered (B) cornea suggest that tissue-selective targeted delivery of decorin gene with AAV5 in the rabbit stroma does not induce changes in corneal structure, thickness or shape. The TEM did not detect significant differences in collagen fibril diameter and arrangement in the naive (C) and decorin-delivered rabbit corneas (D) indicating that localized therapeutic levels of decorin delivery in the cornea does not appear compromise corneal health. dcn = decorin.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198476&req=5

pone-0026432-g008: Representative optical coherence tomography (A and B) and transmission electron microscopy (C and D) images of rabbits receiving AAV5-gfp or AAV5-dcn.Optical coherence tomography was performed in live rabbits on day 10 after the AAV5-gfp or AAV5-dcn application and transmission electron microscopy was performed in corneas collected 14 days after AAV5-gfp or AAV5-dcn application. The optical scans of no decorin-delivered (A) and decorin-delivered (B) cornea suggest that tissue-selective targeted delivery of decorin gene with AAV5 in the rabbit stroma does not induce changes in corneal structure, thickness or shape. The TEM did not detect significant differences in collagen fibril diameter and arrangement in the naive (C) and decorin-delivered rabbit corneas (D) indicating that localized therapeutic levels of decorin delivery in the cornea does not appear compromise corneal health. dcn = decorin.
Mentions: Visual and slit-lamp clinical examination performed in the eyes of live rabbits did not detect any abnormality in non decorin-delivered or decorin-delivered corneas with AAV5 at all tested time points. No haziness, swelling, redness or infection was detected in visual clinical eye examination except in the region where VEGF pellet was implanted. As expected, several blood vessels sprouting from the limbus towards the VEGF pellet were observed in clinical examination. Optical coherence tomography did not detect alteration in corneal structure, thickness or shape of the stroma in decorin-delivered and no decorin-delivered rabbit corneas (Fig 8A and 8B). Furthermore, TEM analysis of naive and decorin-delivered rabbit corneas did not detect any significant differences between the collagen fibril diameter and arrangement in the stroma (Fig 8C and 8D). Slit-lamp, optical coherence tomography and TEM data allowed us to infer that targeted decorin gene transfer into stroma does not affect optical and structural properties of the cornea, and consequently visual function.

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