섬유주세포에서 호모시스테인이 기질금속단백분해효소-9의 발현과 세포고사에 미치는 영향
Effect of Homocysteine on the Expression of Matrix Metalloproteinase-9 and Apoptosis in Trabecular Meshwork Cells
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목적: 망막내피혈관세포에서 기질금속단백분해효소(matrix metalloproteinase, MMP)-9의 활성과 세포고사를 유발하는 것으로 알려진 호모시스테인(homocysteine, Hcy)이 섬유주세포에 미치는 영향을 알아보고자 하였다. 대상과 방법: 섬유주세포를 일차배양한 다음 저농도포도당(5 mM)을 함유한 배지(low glucose, LG)와 고농도(20 mM)의 포도당을 함유한 배지(high glucose, HG)를 이용하여 100 μM Hcy에 3일간 노출시켰다. MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay로 세포의 생존을 측정하였고 western blot으로 MMP-9과 tissue inhibitor of MMP (TIMP)-1 단백의 활성을측정하였다. Annexin-propidium iodide 이중염색을 이용한 유세포분석을 시행하여 세포고사의 정도를 측정하였다. 결과: HG에 Hcy를 노출시킨 경우 HG에 비하여 세포의 생존율이 감소하였다(p=0.036). HG에 비하여 HG와 Hcy를 함께 노출시킨경우 TIMP-1 단백의 활성은 저하되었으나 통계학적으로 유의하지 않았으며(p=0.094), MMP-9 단백의 활성도 유의한 차이를 보이지않았다(p=0.413). 또한 HG에 Hcy를 함께 노출시킨 경우 HG에 비하여 세포고사의 정도에는 영향을 미치지 않았다(p=0.437). 결론: 망막내피혈관세포와는 달리 섬유주세포에서는 Hcy가 TIMP-1과 MMP-9 단백의 활성, 세포고사의 정도에는 유의한 영향을 미치지 않았다. 따라서 섬유주세포에서 Hcy가 미치는 영향은 제한적일 것으로 생각된다.
Abstract
Purpose: In retinal endothelial cells, homocysteine (Hcy) activates matrix metalloproteinase (MMP)-9, which results in apoptosis. This study investigated these effects of Hcy in human trabecular meshwork cells (HTMC). Methods: HTMC cultures using 5 mM low or 20 mM high glucose (HG)-containing media were exposed to 100 μM Hcy for 3 days. Cell viability was assessed with the MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. The MMP-9 and tissue inhibitor of MMP (TIMP)-1 levels were measured by western blotting and the degree of apoptosis was analyzed with flow cytometry using Annexin-propidium iodide double staining. Results: Exposure to Hcy in HG decreased cell viability compared to HG alone (p = 0.036). Compared to HG alone, co-exposure to Hcy with HG decreased the TIMP-1 levels, albeit not significantly (p = 0.094), and did not affect the MMP-9 levels (p = 0.413). In addition, co-exposure to Hcy with HG produced no difference in the degree of apoptosis compared to HG alone (p = 0.437). Conclusions: Unlike in retinal endothelial cells, Hcy did not affect the activities of TIMP-1, MMP-9, or the degrees of apoptosis significantly in HTMC. Thus, the effects Hcy may be limited in HTMC.
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