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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 stereomicroscopy images showing VEGF-induced CNV in no decorin-delivered control (A, C and E) and decorin-delivered (B, D and F) rabbit corneas.Rabbit eyes were imaged early (5 day, Panels A, B), mid (10 day, Panels C, D), and late (14 day, Panels E, F) stages after VEGF pellet implantation. The 100 µl AAV5 viral titer (5×1012 vg/ml) expressing no gene/gfp or decorin was topically applied onto the cornea after removing corneal epithelium for single application for 2 minutes. A statistically significant inhibition of neovascularization was observed at three tested early, mid and late stages of the CNV. Scale bar denotes 2 mm. dcn = decorin.
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pone-0026432-g002: Representative stereomicroscopy images showing VEGF-induced CNV in no decorin-delivered control (A, C and E) and decorin-delivered (B, D and F) rabbit corneas.Rabbit eyes were imaged early (5 day, Panels A, B), mid (10 day, Panels C, D), and late (14 day, Panels E, F) stages after VEGF pellet implantation. The 100 µl AAV5 viral titer (5×1012 vg/ml) expressing no gene/gfp or decorin was topically applied onto the cornea after removing corneal epithelium for single application for 2 minutes. A statistically significant inhibition of neovascularization was observed at three tested early, mid and late stages of the CNV. Scale bar denotes 2 mm. dcn = decorin.

Mentions: Figure 2 shows stereomicroscopic images depicting the area, length and density of VEGF-induced blood vessels in decorin-delivered and no decorin-delivered (control) rabbit corneas observed at 3 different time points. Implanted VEGF produced a strong angiogenic response as evidenced by the presence of blood vessels in avascular regions of the cornea. The peak angiogenic response in control corneas that received AAV5-naked or AAV5-gfp vector was observed on day 10. Decorin-delivered rabbit corneas showed significant decrease in the area, length, thickness and density of the blood vessels at the early- (day 5; Fig 2B), mid- (day 10; Fig 2D) and late- (day 14; Fig 2F) stages of CNV compared to corresponding control (no decorin-delivered) corneas (Fig. 2A, 2C and 2E). To quantify the efficacy of AAV5-mediated decorin gene therapy, area, length and density of blood vessels in the cornea was determined with NIH software Image J. Figure 3 shows mean corneal vasculature area detected in decorin-delivered and control (no decorin-delivered) rabbit corneas at three time points namely 5, 10 and 14 day which represent early, mid and late stages of CNV, respectively. The control corneas (AAV5-naked or AAV5-GFP treated) showed a mean vasculature area of 10 mm2 at day 5, 12.7 mm2 at day 10, and 10.3 mm2 at day 14. Contrary to this, decorin-delivered rabbit corneas showed mean CNV area of 4.8 mm2 on day 5, 4.3 mm2 on day 10, and 3.8 mm2 on day 14. The relative comparison of CNV data between the control and decorin-delivered corneas revealed a statistically significant reduction in corneal neovascularization by 52±5.5% on day 5 (ψ = p<0.05), 66±6.5% on day 10 (* = p<0.001), and 63±6.3% on day 14 (ζ = p<0.01) (Fig. 3).


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 stereomicroscopy images showing VEGF-induced CNV in no decorin-delivered control (A, C and E) and decorin-delivered (B, D and F) rabbit corneas.Rabbit eyes were imaged early (5 day, Panels A, B), mid (10 day, Panels C, D), and late (14 day, Panels E, F) stages after VEGF pellet implantation. The 100 µl AAV5 viral titer (5×1012 vg/ml) expressing no gene/gfp or decorin was topically applied onto the cornea after removing corneal epithelium for single application for 2 minutes. A statistically significant inhibition of neovascularization was observed at three tested early, mid and late stages of the CNV. Scale bar denotes 2 mm. dcn = decorin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198476&req=5

pone-0026432-g002: Representative stereomicroscopy images showing VEGF-induced CNV in no decorin-delivered control (A, C and E) and decorin-delivered (B, D and F) rabbit corneas.Rabbit eyes were imaged early (5 day, Panels A, B), mid (10 day, Panels C, D), and late (14 day, Panels E, F) stages after VEGF pellet implantation. The 100 µl AAV5 viral titer (5×1012 vg/ml) expressing no gene/gfp or decorin was topically applied onto the cornea after removing corneal epithelium for single application for 2 minutes. A statistically significant inhibition of neovascularization was observed at three tested early, mid and late stages of the CNV. Scale bar denotes 2 mm. dcn = decorin.
Mentions: Figure 2 shows stereomicroscopic images depicting the area, length and density of VEGF-induced blood vessels in decorin-delivered and no decorin-delivered (control) rabbit corneas observed at 3 different time points. Implanted VEGF produced a strong angiogenic response as evidenced by the presence of blood vessels in avascular regions of the cornea. The peak angiogenic response in control corneas that received AAV5-naked or AAV5-gfp vector was observed on day 10. Decorin-delivered rabbit corneas showed significant decrease in the area, length, thickness and density of the blood vessels at the early- (day 5; Fig 2B), mid- (day 10; Fig 2D) and late- (day 14; Fig 2F) stages of CNV compared to corresponding control (no decorin-delivered) corneas (Fig. 2A, 2C and 2E). To quantify the efficacy of AAV5-mediated decorin gene therapy, area, length and density of blood vessels in the cornea was determined with NIH software Image J. Figure 3 shows mean corneal vasculature area detected in decorin-delivered and control (no decorin-delivered) rabbit corneas at three time points namely 5, 10 and 14 day which represent early, mid and late stages of CNV, respectively. The control corneas (AAV5-naked or AAV5-GFP treated) showed a mean vasculature area of 10 mm2 at day 5, 12.7 mm2 at day 10, and 10.3 mm2 at day 14. Contrary to this, decorin-delivered rabbit corneas showed mean CNV area of 4.8 mm2 on day 5, 4.3 mm2 on day 10, and 3.8 mm2 on day 14. The relative comparison of CNV data between the control and decorin-delivered corneas revealed a statistically significant reduction in corneal neovascularization by 52±5.5% on day 5 (ψ = p<0.05), 66±6.5% on day 10 (* = p<0.001), and 63±6.3% on day 14 (ζ = p<0.01) (Fig. 3).

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