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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 immunoblot (A) and TUNEL assay (B) of rabbit corneas receiving AAV5-decorin or AAV5-gfp.Representative western blotting showing AAV5-controlled decorin delivery in rabbit corneas collected 14 days after VEGF-implantation (A) and TUNEL assay detecting keratocyte apoptosis in rabbit corneas collected 4 hours after epithelial removal by two different ways (B). A strong band in AAV5-dcn treated rabbit corneas of day-14 time point reveals significant decorin delivery (8.7-fold, p<0.05) in the stroma, and a weak decorin band in naive or AAV5-gfp treated corneas indicates low endogenous decorin expression (A). GAPDH was used to confirm equal protein loading in each well and normalization of the data. TUNEL-stained rabbit corneal sections of 4-hour time point shown in Panel B demonstrate minimal keratocyte apoptosis (arrow) in cornea deepithelialized via gentle scrapping with #64 surgical blade by running blade at 45° angle (left panel) compared to high keratocyte death in cornea in which epithelium was removed in random manner with #64 surgical blade (right panel). Scale bar denotes 100 µm. dcn = decorin.
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pone-0026432-g001: Representative immunoblot (A) and TUNEL assay (B) of rabbit corneas receiving AAV5-decorin or AAV5-gfp.Representative western blotting showing AAV5-controlled decorin delivery in rabbit corneas collected 14 days after VEGF-implantation (A) and TUNEL assay detecting keratocyte apoptosis in rabbit corneas collected 4 hours after epithelial removal by two different ways (B). A strong band in AAV5-dcn treated rabbit corneas of day-14 time point reveals significant decorin delivery (8.7-fold, p<0.05) in the stroma, and a weak decorin band in naive or AAV5-gfp treated corneas indicates low endogenous decorin expression (A). GAPDH was used to confirm equal protein loading in each well and normalization of the data. TUNEL-stained rabbit corneal sections of 4-hour time point shown in Panel B demonstrate minimal keratocyte apoptosis (arrow) in cornea deepithelialized via gentle scrapping with #64 surgical blade by running blade at 45° angle (left panel) compared to high keratocyte death in cornea in which epithelium was removed in random manner with #64 surgical blade (right panel). Scale bar denotes 100 µm. dcn = decorin.

Mentions: The delivery of decorin gene with a single topical application of AAV5-dcn vector in the rabbit cornea was validated with western blotting. Figure 1A shows the levels of detected decorin in naive, control (no decorin) and decorin-delivered rabbit corneas. The protein lysates prepared from the AAV5-dcn treated corneas showed significant 8.7±0.4 (p<0.05) fold higher levels of decorin confirming substantial delivery of therapeutic gene, decorin, in the rabbit corneas with selected AAV serotype. Contrary to this, very weak bands of decorin detected in the naive and naked-AAV5 vector treated rabbit corneas confirmed the low endogenous expression of decorin protein in the rabbit cornea.


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 immunoblot (A) and TUNEL assay (B) of rabbit corneas receiving AAV5-decorin or AAV5-gfp.Representative western blotting showing AAV5-controlled decorin delivery in rabbit corneas collected 14 days after VEGF-implantation (A) and TUNEL assay detecting keratocyte apoptosis in rabbit corneas collected 4 hours after epithelial removal by two different ways (B). A strong band in AAV5-dcn treated rabbit corneas of day-14 time point reveals significant decorin delivery (8.7-fold, p<0.05) in the stroma, and a weak decorin band in naive or AAV5-gfp treated corneas indicates low endogenous decorin expression (A). GAPDH was used to confirm equal protein loading in each well and normalization of the data. TUNEL-stained rabbit corneal sections of 4-hour time point shown in Panel B demonstrate minimal keratocyte apoptosis (arrow) in cornea deepithelialized via gentle scrapping with #64 surgical blade by running blade at 45° angle (left panel) compared to high keratocyte death in cornea in which epithelium was removed in random manner with #64 surgical blade (right panel). Scale bar denotes 100 µm. dcn = decorin.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026432-g001: Representative immunoblot (A) and TUNEL assay (B) of rabbit corneas receiving AAV5-decorin or AAV5-gfp.Representative western blotting showing AAV5-controlled decorin delivery in rabbit corneas collected 14 days after VEGF-implantation (A) and TUNEL assay detecting keratocyte apoptosis in rabbit corneas collected 4 hours after epithelial removal by two different ways (B). A strong band in AAV5-dcn treated rabbit corneas of day-14 time point reveals significant decorin delivery (8.7-fold, p<0.05) in the stroma, and a weak decorin band in naive or AAV5-gfp treated corneas indicates low endogenous decorin expression (A). GAPDH was used to confirm equal protein loading in each well and normalization of the data. TUNEL-stained rabbit corneal sections of 4-hour time point shown in Panel B demonstrate minimal keratocyte apoptosis (arrow) in cornea deepithelialized via gentle scrapping with #64 surgical blade by running blade at 45° angle (left panel) compared to high keratocyte death in cornea in which epithelium was removed in random manner with #64 surgical blade (right panel). Scale bar denotes 100 µm. dcn = decorin.
Mentions: The delivery of decorin gene with a single topical application of AAV5-dcn vector in the rabbit cornea was validated with western blotting. Figure 1A shows the levels of detected decorin in naive, control (no decorin) and decorin-delivered rabbit corneas. The protein lysates prepared from the AAV5-dcn treated corneas showed significant 8.7±0.4 (p<0.05) fold higher levels of decorin confirming substantial delivery of therapeutic gene, decorin, in the rabbit corneas with selected AAV serotype. Contrary to this, very weak bands of decorin detected in the naive and naked-AAV5 vector treated rabbit corneas confirmed the low endogenous expression of decorin protein in the rabbit cornea.

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