Microarray Analysis for Genes Associated with Angiogenesis in Diabetic OLETF Keratocytes

ABSTRACT

The purpose of this study was to identify the differences in angiogenesis gene expression between normal and diabetic keratocytes stimulated with interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha). Primarily cultured normal and diabetic keratocytes were treated with 20 ng/mL of IL-1a and TNF-alpha for 6 hr. cDNA was hybridized to an oligonucleotide microarray. Microarray analysis was used to identify differentially expressed genes that were further evaluated by real-time polymerase chain reaction (RT-PCR). Diabetes keratocytes overexpressed vital components of angiogenesis including Agtr1, and under-expressed components related to the blood vessel maturation, including Dcn. Cytokine-treated diabetic keratocytes differentially expressed components of angiogenesis. OLETF keratocytes after treatment with IL-1alpha and TNF-alpha showed the newly expressed 15 and 14 genes, respectively. Newly and commonly under-expressed five genes followed by treatment with both IL-1alpha and TNF-alpha were also evident. RT-PCR showed results similar to the microarray results. Agtr1 and Itga1 showed an increased expression in diabetic keratocytes compared with normal corneal keratocytes, especially after TNF-alpha treatment. Il6 appeared strong expression after interleukin-1alpha treatment, but showed down expression after TNF-alpha treatment. Further studies to analyze and confirm the significance of the identified angiogenetic genes of diabetes are needed.