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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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CD31 Western blot for naive, AAV5+/−decorin-delivered rabbit corneal tissues collected 10 and 14 days after VEGF-implantation.Panel on left side shows immunoblot and on right side shows quantification of western blotting data collected from corneal tissues of day-10 and day-14. A significant decrease in expression of CD31 on day-10 (62%, p<0.05) and day-14 (66%, p<0.05) was detected in decorin-delivered corneas compared to control corneas suggesting that AAV5-mediated decorin gene therapy is efficient in decreasing CNV. β-actin was used to confirm equal loading of protein in each well and normalization of the data. dcn = decorin.
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pone-0026432-g006: CD31 Western blot for naive, AAV5+/−decorin-delivered rabbit corneal tissues collected 10 and 14 days after VEGF-implantation.Panel on left side shows immunoblot and on right side shows quantification of western blotting data collected from corneal tissues of day-10 and day-14. A significant decrease in expression of CD31 on day-10 (62%, p<0.05) and day-14 (66%, p<0.05) was detected in decorin-delivered corneas compared to control corneas suggesting that AAV5-mediated decorin gene therapy is efficient in decreasing CNV. β-actin was used to confirm equal loading of protein in each well and normalization of the data. dcn = decorin.

Mentions: The efficacy of AAV5-dcn gene therapy was also analyzed by performing western blotting of CD31, a pan-endothelial marker found in blood vessels, platelets, granulocytes, and monocytes that are involved in the formation of new blood vessels. Figure 6 shows results of CD31 western blotting performed with protein lysates prepared from control and decorin-delivered rabbit corneas collected 10 or 14 days after VEGF implantation. As evident from Fig 6, AAV-mediated decorin delivery in rabbit corneas reduced CD31 expression 62±3% (p<0.05) on day-10 and 67±6% (p<0.05) on day14 suggesting that maximal inhibition was attained by day-10 and then continued until the longest tested time point of day-14. The detection of similar intensity β-actin bands confirmed equal loading of protein samples.


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)

CD31 Western blot for naive, AAV5+/−decorin-delivered rabbit corneal tissues collected 10 and 14 days after VEGF-implantation.Panel on left side shows immunoblot and on right side shows quantification of western blotting data collected from corneal tissues of day-10 and day-14. A significant decrease in expression of CD31 on day-10 (62%, p<0.05) and day-14 (66%, p<0.05) was detected in decorin-delivered corneas compared to control corneas suggesting that AAV5-mediated decorin gene therapy is efficient in decreasing CNV. β-actin was used to confirm equal loading of protein in each well and normalization of the data. dcn = decorin.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026432-g006: CD31 Western blot for naive, AAV5+/−decorin-delivered rabbit corneal tissues collected 10 and 14 days after VEGF-implantation.Panel on left side shows immunoblot and on right side shows quantification of western blotting data collected from corneal tissues of day-10 and day-14. A significant decrease in expression of CD31 on day-10 (62%, p<0.05) and day-14 (66%, p<0.05) was detected in decorin-delivered corneas compared to control corneas suggesting that AAV5-mediated decorin gene therapy is efficient in decreasing CNV. β-actin was used to confirm equal loading of protein in each well and normalization of the data. dcn = decorin.
Mentions: The efficacy of AAV5-dcn gene therapy was also analyzed by performing western blotting of CD31, a pan-endothelial marker found in blood vessels, platelets, granulocytes, and monocytes that are involved in the formation of new blood vessels. Figure 6 shows results of CD31 western blotting performed with protein lysates prepared from control and decorin-delivered rabbit corneas collected 10 or 14 days after VEGF implantation. As evident from Fig 6, AAV-mediated decorin delivery in rabbit corneas reduced CD31 expression 62±3% (p<0.05) on day-10 and 67±6% (p<0.05) on day14 suggesting that maximal inhibition was attained by day-10 and then continued until the longest tested time point of day-14. The detection of similar intensity β-actin bands confirmed equal loading of protein samples.

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