Gene Expression Analysis of Antiangiogenic Factors Modified by Transpupillary Thermotherapy.

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16591765
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Ophthalmology
• Research Institution: Saitama Medical School
• Principal Investigator: MORI Keisuke Saitama Medical School, Department of Ophthalmology, Associate Professor, 医学部, 助教授 (90251090)
• Project Period (FY): 2004 – 2005
• Keywords: transpupillary thermotherapy / angiogenesis related genes / age-related macular degeneration / microarray / qRT-PCR

Research Abstract

PURPOSE : To study gene expression changes in the rat retina and choroid following transpupillary thermotherapy (TTT) and to identify molecular mechanisms that may enhance treatment of choroidal neovascularization, complicating age-related macular degeneration.
METHODS : In Brown Norway rats, one fundus was treated with an 810-nm diode laser while the contralateral fundus received no treatment. Six TTT lesions per eye were placed using the following laser settings ; 50 mW, for 60 seconds using a spot size of 3 mm in diameter. The mRNA was extracted from enucleated eyes and was processed for cDNA microarray analysis, in duplicate, by a two-color dye swapping method. Genes with increased expression following duplicate microarray were validated by semi-quantitative reverse transcription PCR and quantitative real-time PCR (qRT-PCR). Additional eyes were studied to evaluate the time course of gene expression changes occurring between 2 and 72 hours following TTT.
RESULTS : Laser-induced lesions were not visible by funduscopy two hours after TTT, indicating that the laser-power used was sub-threshold. Of the 14815 cDNA elements on the array, 12 genes were up-regulated in TTT treated eyes. Up-regulation of 8 of these 12 genes could be verified by semi-quantitative RT-PCR. The eight verified genes were Asns, EPCR, Fgl, IL-1β, MCP-1, MT-2, NMDMC and TSP-1. QRT-PCR analysis demonstrated peak expression of Fgl, MCP-1 and TSP-1 at 2 hours, while Asns, MT-2 and NMDMC peaked at the six hour time point.
CONCLUSION : This study demonstrates up-regulation of angiogenesis and coagulation-related genes following TTT. The response profile and its temporal relationships provide insight into the molecular mechanisms that lead to vascular occlusion and antiangiogenesis induced by TTT.