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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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Quantification of mRNA expression of pro- and anti-angiogenic genes by real-time PCR.Real-time PCR was carried out in naive, control (no decorin-delivered) and decorin-delivered rabbit corneal tissues collected 14 days after VEGF implantation. Decorin gene therapy delivered with AAV5 showed marked decrease in the mRNA expression of VEGF and MCP-1 and less pronounced alteration in angiopoietin and PEDF genes. Our data suggests anti-angiogenic effects of decorin gene therapy are mediated by the down-regulation of pro-angiogeneic (VEGF, MCP1 and angiopoietin) and up-regulation of anti-angiogenic (PEDF) genes. * = p<0.001 compared to naive; ζ = p<0.05 compared to naive; ψ = p<0.05 compared to no decorin (dcn)-delivered.
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pone-0026432-g007: Quantification of mRNA expression of pro- and anti-angiogenic genes by real-time PCR.Real-time PCR was carried out in naive, control (no decorin-delivered) and decorin-delivered rabbit corneal tissues collected 14 days after VEGF implantation. Decorin gene therapy delivered with AAV5 showed marked decrease in the mRNA expression of VEGF and MCP-1 and less pronounced alteration in angiopoietin and PEDF genes. Our data suggests anti-angiogenic effects of decorin gene therapy are mediated by the down-regulation of pro-angiogeneic (VEGF, MCP1 and angiopoietin) and up-regulation of anti-angiogenic (PEDF) genes. * = p<0.001 compared to naive; ζ = p<0.05 compared to naive; ψ = p<0.05 compared to no decorin (dcn)-delivered.

Mentions: To investigate the possible molecular mechanism of decorin gene therapy for inhibiting CNV, the expression of pro-angiogenic factors such as VEGF, MCP1, angiopoietin and anti-angiogenic factors such as PEDF was analyzed in naive, control (no-decorin) and decorin-delivered rabbit corneas collected 14 days after VEGF-implantation. Figure 7 shows the relative change in the expression of VEGF, MCP1, angiopoietin, and PEDF genes in the cDNA sample of the naive, control (no-decorin) and decorin-delivered rabbit corneas. All cDNA samples with VEGF-induced CNV showed statistically significant increase in VEGF and MCP1 and a less pronounced increase in angiopoietin as compared to cDNA samples obtained from naive corneas. The decorin-delivered corneas showed a significant decrease in VEGF expression (5 fold, p<0.05) and MCP1 (3 fold, p<0.05). Contrary to this, decorin-delivered cornea showed lower angiopoietin and mildly increased PEDF expression, however, data was not statistically significant (Fig. 7).


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)

Quantification of mRNA expression of pro- and anti-angiogenic genes by real-time PCR.Real-time PCR was carried out in naive, control (no decorin-delivered) and decorin-delivered rabbit corneal tissues collected 14 days after VEGF implantation. Decorin gene therapy delivered with AAV5 showed marked decrease in the mRNA expression of VEGF and MCP-1 and less pronounced alteration in angiopoietin and PEDF genes. Our data suggests anti-angiogenic effects of decorin gene therapy are mediated by the down-regulation of pro-angiogeneic (VEGF, MCP1 and angiopoietin) and up-regulation of anti-angiogenic (PEDF) genes. * = p<0.001 compared to naive; ζ = p<0.05 compared to naive; ψ = p<0.05 compared to no decorin (dcn)-delivered.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198476&req=5

pone-0026432-g007: Quantification of mRNA expression of pro- and anti-angiogenic genes by real-time PCR.Real-time PCR was carried out in naive, control (no decorin-delivered) and decorin-delivered rabbit corneal tissues collected 14 days after VEGF implantation. Decorin gene therapy delivered with AAV5 showed marked decrease in the mRNA expression of VEGF and MCP-1 and less pronounced alteration in angiopoietin and PEDF genes. Our data suggests anti-angiogenic effects of decorin gene therapy are mediated by the down-regulation of pro-angiogeneic (VEGF, MCP1 and angiopoietin) and up-regulation of anti-angiogenic (PEDF) genes. * = p<0.001 compared to naive; ζ = p<0.05 compared to naive; ψ = p<0.05 compared to no decorin (dcn)-delivered.
Mentions: To investigate the possible molecular mechanism of decorin gene therapy for inhibiting CNV, the expression of pro-angiogenic factors such as VEGF, MCP1, angiopoietin and anti-angiogenic factors such as PEDF was analyzed in naive, control (no-decorin) and decorin-delivered rabbit corneas collected 14 days after VEGF-implantation. Figure 7 shows the relative change in the expression of VEGF, MCP1, angiopoietin, and PEDF genes in the cDNA sample of the naive, control (no-decorin) and decorin-delivered rabbit corneas. All cDNA samples with VEGF-induced CNV showed statistically significant increase in VEGF and MCP1 and a less pronounced increase in angiopoietin as compared to cDNA samples obtained from naive corneas. The decorin-delivered corneas showed a significant decrease in VEGF expression (5 fold, p<0.05) and MCP1 (3 fold, p<0.05). Contrary to this, decorin-delivered cornea showed lower angiopoietin and mildly increased PEDF expression, however, data was not statistically significant (Fig. 7).

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