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A reproducible and quantifiable model of choroidal neovascularization induced by VEGF A165 after subretinal adenoviral gene transfer in the rabbit.

Julien S, Kreppel F, Beck S, Heiduschka P, Brito V, Schnichels S, Kochanek S, Schraermeyer U - Mol. Vis. (2008)

Bottom Line: To determine the effects of the vascular endothelial growth factor (VEGF)-A(165) delivered using a high capacity adenoviral vector (HC Ad.VEGF-A) on vascular growth and pathological changes in the rabbit eye.Our findings present clear indications that there is a significant effect on the endothelial cells of the choriocapillaris after subretinal transduction of the retinal pigment epithelium (RPE) with VEGF-A(165) vector.Many of the features of our experimental CNV resemble those observed clinically in patients having wet age-related macular degeneration.

View Article: PubMed Central - PubMed

Affiliation: Section of Experimental Vitreoretinal Surgery, University Eye Hospital of Tuebingen, Germany. Sylvie.Julien@med.unituebingen.de

ABSTRACT

Purpose: To determine the effects of the vascular endothelial growth factor (VEGF)-A(165) delivered using a high capacity adenoviral vector (HC Ad.VEGF-A) on vascular growth and pathological changes in the rabbit eye. To combine different detection methods of VEGF-A(165) overexpression-induced neovascularization in the rabbit.

Methods: HC Ad.VEGF-A(165) was constructed and injected at 5 x 10(6) infectious units (iu) into the subretinal space of rabbit eyes. Two and four weeks postinjection, the development of neovascularization and the expression of HC Ad-transduced VEGF-A(165) protein were followed up in vivo by scanning laser ophthalmoscopy, fluorescein and indocyanine green angiographies and ex vivo by electron microscopy and immunohistochemistry

Results: We observed a choroidal neovascularization (CNV) with leakage in 83% of the rabbit eyes. Our findings present clear indications that there is a significant effect on the endothelial cells of the choriocapillaris after subretinal transduction of the retinal pigment epithelium (RPE) with VEGF-A(165) vector. The choroidal endothelial cells were activated, adherent junctions opened, and the fenestration was minimized, while the extracellular matrix localized between the RPE and the endothelium of the choriocapillaris was enlarged toward the lumen of the vessels, inducing a deep invagination of the endothelial cells into the vessel lumen. They also proliferated and formed pathological vessels in the subretinal space. Moreover,there was an increased expression of basic fibroblast growth factor and VEGF-A accompanied by macrophage stimulation, retinal edema, and photoreceptor loss.

Conclusions: This is the first model of VEGF-induced CNV in the rabbit in which the pathological events following overexpression of VEGF by RPE cells have been described in detail. Many of the features of our experimental CNV resemble those observed clinically in patients having wet age-related macular degeneration.

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Immunohistochemical findings after transduction with HC Ad.VEGF-A with hematoxylin and eosin counterstain. A: Staining with tomato lectin showed that most cells penetrating the disrupted retinal pigment epithelium (RPE) cell layer were of endothelial origin (red). Inner and outer retinal nuclear layers were mixed. Single RPE cells (black arrow) were surrounded by proliferating endothelial cells. B: VEGF-A was highly expressed in the retinal scar (white asterisk) and in the RPE close to the retinal scar (black asterisk) but this expression rapidly decreased the farther it was from the retinal scar (E). The expression of bFGF was not as strong in cells of choroidal origin than in retinal cells (C). D: Proliferating cells at the retinal choroidal interface were immunoreactive for Ki67 (black arrowheads). The inset in (D) demonstrates the endothelial nature of dividing cells by double labeling for Ki67 (red) and tomato lectin (green). F: Albumin (red) was present in the choroid and the fiber matrix at the vitreoretinal interface. It was absent in the unaffected retina (black asterisk). Two selected areas are shown enlarged (G, H). Choroidal retinal scarring and leakage of albumin are visible in one of the enlarged areas (G). Albumin was not localized within proliferating cells surrounding single RPE cells (black arrows). This may be why ICG did not enter such sites (see Figure 2I,M). H: The second enlarged area revealed albumin leakage within the retina that, as shown in this panel, was not fused with RPE or choroid (Ch). Abbreviations: INL, inner nuclear layer; GCL, ganglion cell layer.
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f6: Immunohistochemical findings after transduction with HC Ad.VEGF-A with hematoxylin and eosin counterstain. A: Staining with tomato lectin showed that most cells penetrating the disrupted retinal pigment epithelium (RPE) cell layer were of endothelial origin (red). Inner and outer retinal nuclear layers were mixed. Single RPE cells (black arrow) were surrounded by proliferating endothelial cells. B: VEGF-A was highly expressed in the retinal scar (white asterisk) and in the RPE close to the retinal scar (black asterisk) but this expression rapidly decreased the farther it was from the retinal scar (E). The expression of bFGF was not as strong in cells of choroidal origin than in retinal cells (C). D: Proliferating cells at the retinal choroidal interface were immunoreactive for Ki67 (black arrowheads). The inset in (D) demonstrates the endothelial nature of dividing cells by double labeling for Ki67 (red) and tomato lectin (green). F: Albumin (red) was present in the choroid and the fiber matrix at the vitreoretinal interface. It was absent in the unaffected retina (black asterisk). Two selected areas are shown enlarged (G, H). Choroidal retinal scarring and leakage of albumin are visible in one of the enlarged areas (G). Albumin was not localized within proliferating cells surrounding single RPE cells (black arrows). This may be why ICG did not enter such sites (see Figure 2I,M). H: The second enlarged area revealed albumin leakage within the retina that, as shown in this panel, was not fused with RPE or choroid (Ch). Abbreviations: INL, inner nuclear layer; GCL, ganglion cell layer.

Mentions: Semithin sections of the eye shown in Figure 2J,K. A: The endothelial cell layer of the choriocapillaris was irregular, and endothelial cells protruded into the vessel lumen (black arrowheads). Evidence for this is presented by electron microscopy (see Figure 5A-C). The retinal pigment epithelium (RPE) cell layer was disrupted (white arrowheads), and endothelial cells migrated and proliferated into the subretinal space or between Bruch’s membrane and RPE (inset). Evidence that these cells were endothelial cells is presented by immunohistochemistry (see Figure 6A). These cellular proliferations were either solid (white asterisk in A) or loosely packed with interstitial spaces (black asterisk in B). The photoreceptors have already degenerated, and retinal scar was closely connected to the RPE and proliferating cells. This was probably why fluorescein leakage was restricted to the spotted roundish areas visible in Figure 2. An immature capillary containing an erythrocyte was located distally to the RPE (B, white arrow). The melanocytes of the choroids were located below the deeper choroidal vessels and are not shown. C and D: After injection of HC Ad. EGFP or PBS, the RPE, choriocapillaris (arrowheads) and deeper choroidal vessels (arrow) appeared to be normal. The pigmented layer, consisting predominately of melanocytes, is marked by a black asterisk. The double arrows in A-C indicate growth of extracellular matrix and vessel layers of the choroid after VEGF expression (A, B) compared to (C).


A reproducible and quantifiable model of choroidal neovascularization induced by VEGF A165 after subretinal adenoviral gene transfer in the rabbit.

Julien S, Kreppel F, Beck S, Heiduschka P, Brito V, Schnichels S, Kochanek S, Schraermeyer U - Mol. Vis. (2008)

Immunohistochemical findings after transduction with HC Ad.VEGF-A with hematoxylin and eosin counterstain. A: Staining with tomato lectin showed that most cells penetrating the disrupted retinal pigment epithelium (RPE) cell layer were of endothelial origin (red). Inner and outer retinal nuclear layers were mixed. Single RPE cells (black arrow) were surrounded by proliferating endothelial cells. B: VEGF-A was highly expressed in the retinal scar (white asterisk) and in the RPE close to the retinal scar (black asterisk) but this expression rapidly decreased the farther it was from the retinal scar (E). The expression of bFGF was not as strong in cells of choroidal origin than in retinal cells (C). D: Proliferating cells at the retinal choroidal interface were immunoreactive for Ki67 (black arrowheads). The inset in (D) demonstrates the endothelial nature of dividing cells by double labeling for Ki67 (red) and tomato lectin (green). F: Albumin (red) was present in the choroid and the fiber matrix at the vitreoretinal interface. It was absent in the unaffected retina (black asterisk). Two selected areas are shown enlarged (G, H). Choroidal retinal scarring and leakage of albumin are visible in one of the enlarged areas (G). Albumin was not localized within proliferating cells surrounding single RPE cells (black arrows). This may be why ICG did not enter such sites (see Figure 2I,M). H: The second enlarged area revealed albumin leakage within the retina that, as shown in this panel, was not fused with RPE or choroid (Ch). Abbreviations: INL, inner nuclear layer; GCL, ganglion cell layer.
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f6: Immunohistochemical findings after transduction with HC Ad.VEGF-A with hematoxylin and eosin counterstain. A: Staining with tomato lectin showed that most cells penetrating the disrupted retinal pigment epithelium (RPE) cell layer were of endothelial origin (red). Inner and outer retinal nuclear layers were mixed. Single RPE cells (black arrow) were surrounded by proliferating endothelial cells. B: VEGF-A was highly expressed in the retinal scar (white asterisk) and in the RPE close to the retinal scar (black asterisk) but this expression rapidly decreased the farther it was from the retinal scar (E). The expression of bFGF was not as strong in cells of choroidal origin than in retinal cells (C). D: Proliferating cells at the retinal choroidal interface were immunoreactive for Ki67 (black arrowheads). The inset in (D) demonstrates the endothelial nature of dividing cells by double labeling for Ki67 (red) and tomato lectin (green). F: Albumin (red) was present in the choroid and the fiber matrix at the vitreoretinal interface. It was absent in the unaffected retina (black asterisk). Two selected areas are shown enlarged (G, H). Choroidal retinal scarring and leakage of albumin are visible in one of the enlarged areas (G). Albumin was not localized within proliferating cells surrounding single RPE cells (black arrows). This may be why ICG did not enter such sites (see Figure 2I,M). H: The second enlarged area revealed albumin leakage within the retina that, as shown in this panel, was not fused with RPE or choroid (Ch). Abbreviations: INL, inner nuclear layer; GCL, ganglion cell layer.
Mentions: Semithin sections of the eye shown in Figure 2J,K. A: The endothelial cell layer of the choriocapillaris was irregular, and endothelial cells protruded into the vessel lumen (black arrowheads). Evidence for this is presented by electron microscopy (see Figure 5A-C). The retinal pigment epithelium (RPE) cell layer was disrupted (white arrowheads), and endothelial cells migrated and proliferated into the subretinal space or between Bruch’s membrane and RPE (inset). Evidence that these cells were endothelial cells is presented by immunohistochemistry (see Figure 6A). These cellular proliferations were either solid (white asterisk in A) or loosely packed with interstitial spaces (black asterisk in B). The photoreceptors have already degenerated, and retinal scar was closely connected to the RPE and proliferating cells. This was probably why fluorescein leakage was restricted to the spotted roundish areas visible in Figure 2. An immature capillary containing an erythrocyte was located distally to the RPE (B, white arrow). The melanocytes of the choroids were located below the deeper choroidal vessels and are not shown. C and D: After injection of HC Ad. EGFP or PBS, the RPE, choriocapillaris (arrowheads) and deeper choroidal vessels (arrow) appeared to be normal. The pigmented layer, consisting predominately of melanocytes, is marked by a black asterisk. The double arrows in A-C indicate growth of extracellular matrix and vessel layers of the choroid after VEGF expression (A, B) compared to (C).

Bottom Line: To determine the effects of the vascular endothelial growth factor (VEGF)-A(165) delivered using a high capacity adenoviral vector (HC Ad.VEGF-A) on vascular growth and pathological changes in the rabbit eye.Our findings present clear indications that there is a significant effect on the endothelial cells of the choriocapillaris after subretinal transduction of the retinal pigment epithelium (RPE) with VEGF-A(165) vector.Many of the features of our experimental CNV resemble those observed clinically in patients having wet age-related macular degeneration.

View Article: PubMed Central - PubMed

Affiliation: Section of Experimental Vitreoretinal Surgery, University Eye Hospital of Tuebingen, Germany. Sylvie.Julien@med.unituebingen.de

ABSTRACT

Purpose: To determine the effects of the vascular endothelial growth factor (VEGF)-A(165) delivered using a high capacity adenoviral vector (HC Ad.VEGF-A) on vascular growth and pathological changes in the rabbit eye. To combine different detection methods of VEGF-A(165) overexpression-induced neovascularization in the rabbit.

Methods: HC Ad.VEGF-A(165) was constructed and injected at 5 x 10(6) infectious units (iu) into the subretinal space of rabbit eyes. Two and four weeks postinjection, the development of neovascularization and the expression of HC Ad-transduced VEGF-A(165) protein were followed up in vivo by scanning laser ophthalmoscopy, fluorescein and indocyanine green angiographies and ex vivo by electron microscopy and immunohistochemistry

Results: We observed a choroidal neovascularization (CNV) with leakage in 83% of the rabbit eyes. Our findings present clear indications that there is a significant effect on the endothelial cells of the choriocapillaris after subretinal transduction of the retinal pigment epithelium (RPE) with VEGF-A(165) vector. The choroidal endothelial cells were activated, adherent junctions opened, and the fenestration was minimized, while the extracellular matrix localized between the RPE and the endothelium of the choriocapillaris was enlarged toward the lumen of the vessels, inducing a deep invagination of the endothelial cells into the vessel lumen. They also proliferated and formed pathological vessels in the subretinal space. Moreover,there was an increased expression of basic fibroblast growth factor and VEGF-A accompanied by macrophage stimulation, retinal edema, and photoreceptor loss.

Conclusions: This is the first model of VEGF-induced CNV in the rabbit in which the pathological events following overexpression of VEGF by RPE cells have been described in detail. Many of the features of our experimental CNV resemble those observed clinically in patients having wet age-related macular degeneration.

Show MeSH
Related in: MedlinePlus