Phenotype of transgenic mice overexpressed with inducible nitric oxide synthase in the retina.
BACKGROUND
Unlike its constitutive isoforms, including neuronal and endothelial nitric oxide synthase, inducible nitric oxide synthase (iNOS) along with a series of cytokines are generated in inflammatory pathologic conditions in retinal photoreceptors. In this study, we constructed transgenic mice overexpressing iNOS in the retina to evaluate the effect of sustained, intense iNOS generation in the photoreceptor damage.


METHODS
For construction of opsin/iNOS transgene in the CMVSport 6 expression vector, the 4.4 kb Acc65I/Xhol mouse rod opsin promoter was ligated upstream to a 4.1 kb fragment encoding the complete mouse cDNA of iNOS. From the four founders identified, two heterozygote lines and one homozygote line were established. The presence of iNOS in the retina was confirmed and the pathologic role of iNOS was assessed by detecting nitrotyrosine products and apoptosis. Commercial TUNEL kit was used to detect DNA strand breaks, a later step in a sequence of morphologic changes of apoptosis process.


RESULTS
The insertion and translation of iNOS gene were demonstrated by an intense single 130 kDa band in Western blot and specific immunolocalization at the photoreceptors of the retina. Cellular toxicity in the retinas of transgenic animals was detected by a post-translational modification product, tyrosine-nitrated protein, the most significant one of which was nitrated cytochrome c. Following the accumulation of nitrated mitochondrial proteins and cytochrome c release, marked apoptosis was detected in the photoreceptor cell nuclei of the retina.


CONCLUSIONS
We have generated a pathologic phenotype with sustained iNOS overexpression and, therefore, high output of nitric oxide. Under basal conditions, such overexpression of iNOS causes marked mitochondrial cytochrome c nitration and release and subsequent photoreceptor apoptosis in the retina. Therefore, the modulation of pathways leading to iNOS generation or its effective neutralization can be of significant therapeutic benefit in the oxidative stress-mediated retinal degeneration, a leading cause of blindness.
Department of Ophthalmology, Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Guey Shuang
GS
Wu
Meisheng
M
Jiang
Yi-Hsin
YH
Liu
Yoshiko
Y
Nagaoka
Narsing A
NA
Rao
D000818:Animals; D017209:Apoptosis; D016207:Cytokines / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005838:Genotype; D007249:Inflammation; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D055355:Opsins / Q000378:metabolism; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012160:Retina / Q000201:enzymology*; D019076:Transgenes
D016207:Cytokines; D018076:DNA, Complementary; D055355:Opsins; D052247:Nitric Oxide Synthase Type II