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. 2007 Mar 27:13:457-69.

Adenoviral gene transfer of bioactive TGFbeta1 to the rodent eye as a novel model for anterior subcapsular cataract

Jennifer V Robertson  1 , Zahra Nathu, Anas Najjar, Dhruva Dwivedi, Jack Gauldie, Judith A West-Mays
Affiliations

Adenoviral gene transfer of bioactive TGFbeta1 to the rodent eye as a novel model for anterior subcapsular cataract

Jennifer V Robertson et al. Mol Vis. .

Abstract

Purpose: To produce a gene-transfer model of rodent anterior subcapsular cataracts (ASC) using a replication-deficient, adenoviral vector containing active TGFbeta1. Establishment of this model will be important for further investigations of TGFbeta-induced signaling cascades in ASC.

Methods: Adenovirus containing the transgene for active TGFbeta1 (AdTGFbeta1), beta-galactosidase (AdLacZ), green fluorescent protein (AdGFP) or no transgene (AdDL) was injected into the anterior chamber of C57Bl/6, Smad3 WT and Smad3 KO mice. Four and 21 days after injection, animals were enucleated and eyes were processed and examined by routine histology. Immunolocalization of markers indicative of epithelial to mesenchymal transition (EMT), fibrosis, proliferation and apoptosis was also carried out.

Results: By day 4, treatment with AdLacZ demonstrated transgene expression in multiple structures of the anterior chamber including the lens epithelium. In contrast to AdDL, treatment with AdTGFbeta1 produced alphaSMA-positive subcapsular plaques in all three groups of mice, which shared features reminiscent of human ASC. At day 21, plaques remained alphaSMA-positive and extensive extracellular matrix deposition was observed. The AdTGFbeta1 model was further employed in Smad3 deficient mice and this resulted in the development of small ASC.

Conclusions: Gene transfer of active TGFbeta1 using an adenoviral vector produced cataractous plaques four days postinjection, which were found to develop independent of functional Smad3.

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Figures

Figure 1
Figure 1
Expression of adenovirally transferred LacZ and GFP. Animals were injected with AdLacZ (A-C), AdDL (D), or AdGFP (E) intracamerally. Four days post injection eyes were removed and wholemount stained for LacZ activity (A) followed by paraffin sectioning (B,C,D) or frozen sectioning and visualized using a GFP filter (E). Blue corneal opacity (asterisks in A) indicates that cellular infection and production of transgene was successful. AdDL eyes remained clear (hash marks in A). B and C demonstrate transgene expression in the corneal endothelium (En), iris (Ir), ciliary body (Cb), trabecular meshwork (Tm), and lens epithelium (Ep). Transgene was absent in control vector treated eyes (D). Transgene expression is confirmed in the lens epithelium (Ep) using AdGFP (green). The scale bars in A = 1 mm, in B-D = 25 μm, and in E = 50 μm.
Figure 2
Figure 2
Effects of adenoviral gene transfer of active TGFβ1 after four days. Histological sections from C57 mice injected with AdTGFβ1 (A and B) or AdDL (C and D). Sections were stained with H&E (A and C) or subjected to immunostaining for αSMA (B and D). Focal multilayering occurs in animals treated with AdTGFβ and is associated with induction of αSMA (A, B) in contrast to AdDL treated eyes (C, D). The green stain is αSMA and the blue stain is DAPI. The scale bar is equal to 25 μm.
Figure 3
Figure 3
Immunolocalization of TGFβ1. Naïve (A), AdDL injected (B), and AdTGFβ1 injected eyes (C and D) were sectioned and subjected to TGFβ1 immunohistochemistry at 0 (A), 4 (B and C) and 21 (D) days after injection. Both naïve and AdDL eyes show no immunolocalization of TGFβ1 in the lens epithelium. Animals injected with AdTGFβ1 demonstrate prominent expression of TGFβ1 in the lens epithelium. The green stain is TGFβ1 and the blue stain is DAPI. The scale bar is equal to 25 μm.
Figure 4
Figure 4
Effects of adenoviral gene transfer of active TGFβ1 after twenty one days. Histological sections from C57 mice injected with AdTGFβ1 (A and B) or AdDL (C and D). Sections were stained with H&E (A and C) or subjected to immunostaining for αSMA (B and D). At 21 days post injection AdTGFβ1 treated eyes demonstrate large plaques which express a considerable amount of αSMA in contrast to AdDL treated eyes. The green stain is αSMA and the blue stain is DAPI. The scale bar is equal to 25 μm.
Figure 5
Figure 5
Crystallin expression in AdTGFβ1 treated eyes. Sections were stained with anti-β (A-D; red) or anti-γ-crystallin (E and F; green) antibodies and counterstained with DAPI (blue). Additionally, β-crystallin stained sections were colocalized with αSMA (A-D; green). Sections were taken at four days (A and B) and at twenty one days (C-F) of both AdTGFβ1 (A, C, and E) and AdDL (B, D, and F). Both β- and γ-crystallin expression can be seen with in the plaques of AdTGFβ1 but not AdDL treated eyes. Epithelia of AdDL treated eyes showed no presence of crystallin or αSMA expression. The scale bar is equal to 25 μm.
Figure 6
Figure 6
Matrix staining of AdTGFβ injected eyes. Sections of AdTGFβ1 (A, C, E, and G) and AdDL (B, D, F, and H) lenses taken on days 4 (A, B, E, and F) and 21 (C, D, G, and H) were stained with PAS (A-D) to detect carbohydrates (purple) and Masson's trichrome (E-H) to detect collagens (green). AdTGFβ1 treated lenses showed accumulation of matrix which was barely detectable on day 4, but prominent on day 21. In contrast, AdDL treated lenses showed no matrix accumulation at any time point. The scale bar is equal to 25 μm.
Figure 7
Figure 7
Collagen IV expression. Immunolocalization of collagen IV was performed on paraffin sections of AdTGFβ1 (A and C) and AdDL (B and D) lenses on days 4 (A and B) and 21 (C and D). AdTGFβ1 treated eyes showed a marked accumulation of collagen IV in the plaques which was absent in epithelia of AdDL treated eyes. The red staining is collagen IV and the blue staining is DAPI. The scale bar is equal to 25 μm.
Figure 8
Figure 8
Effects of adenoviral gene transfer of active TGFβ1 to Smad3KO eyes after four days. Histological sections from Smad3KO (A, C, E, and G) and WT (B, D, F, and H) treated with AdTGFβ1. Both groups developed αSMA (brown; C and D) expressing plaques, and showed no difference in cellular proliferation by the PCNA (green) stain (E and F). Smad3 KO (G) animals demonstrated more TUNEL (green) positive cells (arrows) compared to WT (H). The scale bar is equal to 25 μm.
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