Abstract
Abstract: :
Purpose: To evaluate the effects of genetic deletion of Pigment Epithelium-Derived Factor (PEDF), a putative endogenous inhibitor of angiogenesis, on the development of the vasculature of the eye. Methods: A parental BAC (bacterial artificial chromosome) clone was used to construct a large PEDF gene targeting vector, and a lacZ reporter cassette was inserted at the PEDF ATG codon to replace the entire PEDF gene. Eyes were collected at 12 weeks of age from animals homozygous (-/-) or heterozygous (+/-) for the PEDF deletion / lacZ substitution and from wild type (+/+) littermates. One eye from each animal was fixed in 2% paraformaldehyde and processed for visualization of ß-galactosidase (ß-gal) activity. Series of frozen sections were then cut and immunostained for PECAM to visualize vascular endothelial cells. The remaining eye from each animal was embedded in paraffin and sections stained with Hematoxylin and Eosin. In addition, corneas and retinas obtained from animals of the same age and genotype were flat-mounted and stained with fluoresceinated lectin (griffonia simplicifolia, B4) to evaluate vascular patterning and morphology. Results: PEDF knock-out mice appeared healthy from birth, and exhibited no overt developmental phenotype. The corneal and retinal vasculature of adult PEDF (-/-) mice did not show any appreciable abnormality when compared with their wild type (+/+) or heterozygous (+/-) littermates. Moreover, the expression of the lacZ marker gene driven off the PEDF promoter was not distributed in a manner that discretely defined vascular boundaries in the eye. For example, ß-gal positive cells were observed not only in the retinal pigment epithelium, but also widely distributed in the choroid. Similarly, ß-gal staining was evident not only in the cornea (excluding the corneal epithelium cells), but also in the sclera. Moreover, the lacZ expression pattern bore no specific or consistent relationship to tissue-specific vascular boundaries in other areas of the body. Conclusions: Genetic deletion of PEDF does not significantly alter the normal development and patterning of the ocular vasculature, indicating that PEDF does not play an indispensable role as an endogenous inhibitor of angiogenesis and is not responsible for the normal compartmentalization of the ocular vasculature during development.
Keywords: transgenics/knock-outs • retinal pigment epithelium • cornea: basic science