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N.D. Wangsa-Wirawan, R. Kroes, H. Yamamoto, J. Moskal, R.A. Linsenmeier; Hypoxia Inducible Factor 1 alpha(HIF-1) in Cat Retina . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4572.
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Purpose: To investigate the expression of HIF-1α in cat retina under normoxia and hypoxia. Protein levels of HIF-1α in mouse retina have been previously studied. Here, we attempt to investigate the mRNA levels of HIF-1α in cat retina. A large animal model (cat) was used since it has advantages for combining physiological and molecular biological studies. Methods: Northern blots were used to detect expression level of HIF-1α and other genes that are regulated by HIF-1α. Northern blot probes (400-500 nucleotides) were synthesized using PCR with primers derived from corresponding human gene sequences and cDNA templates derived from cat brain. PCR products were then subcloned into E coli and cultured to multiply PCR products. DNA sequencing was performed to verify PRC products. Cat retinas were isolated from cats breathing air (normoxia) and 7-10% oxygen (hypoxia). RNA was isolated from retinas and hybridized with probes in Northern Blots to detect their levels. Results: As expected, primers derived from human gene sequences worked well using PCR with a cat brain-derived cDNA template. Cat genome data is very limited. Hence, primers were derived from corresponding human gene sequences in areas with high homology across various species. A partial cDNA sequence of the cat HIF-1α gene was obtained to use as a probe and was confirmed by DNA sequencing. In the regions sequenced, cat HIF-1α has 95% homology with human HIF-1α. Regulation of various genes involved in angiogenesis, proliferation, survival, and glycolysis in cancerous tissues by HIF-1α has been reported. It is possible that this regulation takes place in the retina as well in a similar fashion. Hence, we are investigating the expression levels of a number genes (IGF-2, VEGF, NOS-2, GLUT-3, LDHA, CA-14, EPO, and EPOR) under normoxia and hypoxia. Conclusions: HIF-1α was expressed in cat retina and its nucleotide sequence has high homology with other mammalian HIFs. Thus, the probes developed will allow the investigation of HIF regulation in normoxia and hypoxia of various durations and severities.
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