Limits...
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.

Show MeSH

Related in: MedlinePlus

Representative images showing CNV in rabbit corneas treated AAV5+/−decorin.Lectin (A–F) and collagen type IV (G–L) immunostaining was carried out in corneal tissue sections obtained from AAV5-gfp and AAV5-dcn treated rabbit corneas collected 14 days after VEGF implantation. As expected very high lectin (A–C) and collagen type IV (G–I) immunostaining was detected in the corneal sections of AAV5-gfp-treated (no decorin-delivered) corneas due CNV induced by the VEGF implantation. The AAV5-mediated decorin gene therapy significantly reduced (p<0.01) the CNV as evident from the markedly less lectin+ (D–F) and collagen type IV+ (J–L) cells in decorin-delivered rabbit corneal sections. DAPI stained nuclei are shown in blue whereas lectin and collagen type IV+ cells in red. Scale bar denotes 100 µm. dcn = decorin.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198476&req=5

pone-0026432-g005: Representative images showing CNV in rabbit corneas treated AAV5+/−decorin.Lectin (A–F) and collagen type IV (G–L) immunostaining was carried out in corneal tissue sections obtained from AAV5-gfp and AAV5-dcn treated rabbit corneas collected 14 days after VEGF implantation. As expected very high lectin (A–C) and collagen type IV (G–I) immunostaining was detected in the corneal sections of AAV5-gfp-treated (no decorin-delivered) corneas due CNV induced by the VEGF implantation. The AAV5-mediated decorin gene therapy significantly reduced (p<0.01) the CNV as evident from the markedly less lectin+ (D–F) and collagen type IV+ (J–L) cells in decorin-delivered rabbit corneal sections. DAPI stained nuclei are shown in blue whereas lectin and collagen type IV+ cells in red. Scale bar denotes 100 µm. dcn = decorin.

Mentions: The inhibitory effects of decorin gene therapy on CNV were further confirmed by performing immunostaining of angiogenesis markers in the tissue sections of rabbit corneas collected 14 days after VEGF-pellet implantation. Tomato lectin specifically stains blood vessels by binding to the components of basement membrane whereas collagen type IV is a component of basal lamina of blood vessels and CD31 is another endothelial marker. Figure 5 shows the results of lectin (Figs. 5A–F) and collagen type IV (Figs. 5G–L) immunostaining performed in no-decorin (AAV5-gfp-treated) and decorin-delivered (AAV5-dcn-treated) rabbit corneas collected on day-14. Significantly less lectin+ (Fig 5E and 5F; 6,859±393 pixels/200x magnification, p<0.01) and collagen type IV+ (Fig 5K and 5L; 1,983±187 pixels/200x magnification, p<0.01) cells were detected in decorin-delivered rabbit corneas compared to no decorin-delivered control corneas stained for lectin (Fig 5B and 5C; 26,608±713 pixels/200x magnification) or collagen type IV (Fig 5H and 5I; 6,646±259 pixels/200x magnification). The digital quantification of lectin+ and collagen type IV+ cells detected 59–65% (p<0.01) decrease in VEGF-induced corneal vasculature by AAV5-dcn gene transfer in rabbits in vivo. Immunostaining using another endothelial marker, CD31, showed similar results (data not shown). These immunohistochemistry investigations suggest that AAV5-mediated decorin gene therapy is highly effective in reducing neovascularization in the rabbit experimental model of CNV.


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 images showing CNV in rabbit corneas treated AAV5+/−decorin.Lectin (A–F) and collagen type IV (G–L) immunostaining was carried out in corneal tissue sections obtained from AAV5-gfp and AAV5-dcn treated rabbit corneas collected 14 days after VEGF implantation. As expected very high lectin (A–C) and collagen type IV (G–I) immunostaining was detected in the corneal sections of AAV5-gfp-treated (no decorin-delivered) corneas due CNV induced by the VEGF implantation. The AAV5-mediated decorin gene therapy significantly reduced (p<0.01) the CNV as evident from the markedly less lectin+ (D–F) and collagen type IV+ (J–L) cells in decorin-delivered rabbit corneal sections. DAPI stained nuclei are shown in blue whereas lectin and collagen type IV+ cells in red. Scale bar denotes 100 µm. dcn = decorin.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026432-g005: Representative images showing CNV in rabbit corneas treated AAV5+/−decorin.Lectin (A–F) and collagen type IV (G–L) immunostaining was carried out in corneal tissue sections obtained from AAV5-gfp and AAV5-dcn treated rabbit corneas collected 14 days after VEGF implantation. As expected very high lectin (A–C) and collagen type IV (G–I) immunostaining was detected in the corneal sections of AAV5-gfp-treated (no decorin-delivered) corneas due CNV induced by the VEGF implantation. The AAV5-mediated decorin gene therapy significantly reduced (p<0.01) the CNV as evident from the markedly less lectin+ (D–F) and collagen type IV+ (J–L) cells in decorin-delivered rabbit corneal sections. DAPI stained nuclei are shown in blue whereas lectin and collagen type IV+ cells in red. Scale bar denotes 100 µm. dcn = decorin.
Mentions: The inhibitory effects of decorin gene therapy on CNV were further confirmed by performing immunostaining of angiogenesis markers in the tissue sections of rabbit corneas collected 14 days after VEGF-pellet implantation. Tomato lectin specifically stains blood vessels by binding to the components of basement membrane whereas collagen type IV is a component of basal lamina of blood vessels and CD31 is another endothelial marker. Figure 5 shows the results of lectin (Figs. 5A–F) and collagen type IV (Figs. 5G–L) immunostaining performed in no-decorin (AAV5-gfp-treated) and decorin-delivered (AAV5-dcn-treated) rabbit corneas collected on day-14. Significantly less lectin+ (Fig 5E and 5F; 6,859±393 pixels/200x magnification, p<0.01) and collagen type IV+ (Fig 5K and 5L; 1,983±187 pixels/200x magnification, p<0.01) cells were detected in decorin-delivered rabbit corneas compared to no decorin-delivered control corneas stained for lectin (Fig 5B and 5C; 26,608±713 pixels/200x magnification) or collagen type IV (Fig 5H and 5I; 6,646±259 pixels/200x magnification). The digital quantification of lectin+ and collagen type IV+ cells detected 59–65% (p<0.01) decrease in VEGF-induced corneal vasculature by AAV5-dcn gene transfer in rabbits in vivo. Immunostaining using another endothelial marker, CD31, showed similar results (data not shown). These immunohistochemistry investigations suggest that AAV5-mediated decorin gene therapy is highly effective in reducing neovascularization in the rabbit experimental model of CNV.

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