Department of Ophthalmology, King's College London, St. Thomas' Hospital, London, United Kingdom.
Invest Ophthalmol Vis Sci. 2010 May;51(5):2363-71. doi: 10.1167/iovs.09-4259. Epub 2009 Dec 10.
The structural and functional demise of aging Bruch's membrane is associated with a reduction in the activity of the matrix metalloproteinase (MMP) degradation system. The gelatinase component of the MMP system consists of MMP2 and MMP9 and two high molecular-weight (HMW1, HMW2) species that are yet to be characterized and whose roles in the aging process are yet to be elucidated. The purpose of this study was to determine the age-related changes in levels of expression and subunit characterization of the HMW gelatinase species of Bruch's membrane.
Gelatin zymography followed by densitometric scanning was used to quantify the level of the HMW species present. Gel-filtration chromatography allowed the fractionation of the gelatinases according to their molecular weight, and subsequent degradation of the HMW species with a mino-phenyl acetate activation, reduction, and alkylation produced subunit fragments for analysis.
Most of the HMW1 and HMW2 pool (80% and 87%, respectively) were tightly bound to the matrix. Aging was associated with significant increases in the levels of HMW1 and HMW2 (P < 0.005 and P < 0.05 respectively). On gel filtration, a single large macromolecular complex (LMMC) was observed containing HMW1, HMW2, MMP9, and some MMP2. Activation-mediated fragmentation of HMW1 and HMW2 showed them to be composed of heteropolymers of MMP2 and MMP9.
The age-related increase of HMW1 and HMW2, together with the formation of LMMC, resulted in the sequestration of MMP2 and MMP9, thereby reducing the free pool for activation. This is likely to contribute to reduced matrix degradation and turnover of Bruch's membrane in both normal aging and age-related macular degeneration.
衰老的布鲁赫膜的结构和功能丧失与基质金属蛋白酶（MMP）降解系统活性降低有关。MMP 系统的明胶酶成分包括 MMP2 和 MMP9 以及两种尚未被表征的高分子量（HMW1、HMW2）物质，其在衰老过程中的作用尚未阐明。本研究的目的是确定布鲁赫膜 HMW 明胶酶物质表达水平的年龄相关性变化及其亚基特征。
使用明胶酶谱法结合密度扫描法定量测定 HMW 物质的水平。凝胶过滤色谱法可根据分子量对明胶酶进行分级，随后用小苯甲酰乙酸激活、还原和烷基化处理 HMW 物质，产生亚基片段进行分析。
大多数 HMW1 和 HMW2 池（分别为 80%和 87%）与基质紧密结合。衰老与 HMW1 和 HMW2 水平的显著增加有关（分别为 P<0.005 和 P<0.05）。在凝胶过滤中，观察到一个包含 HMW1、HMW2、MMP9 和一些 MMP2 的单一大分子复合物（LMMC）。通过激活介导的 HMW1 和 HMW2 的片段化显示它们由 MMP2 和 MMP9 的异聚体组成。
HMW1 和 HMW2 的年龄相关性增加，以及 LMMC 的形成，导致 MMP2 和 MMP9 的隔离，从而减少了可激活的游离池。这可能导致布鲁赫膜在正常衰老和年龄相关性黄斑变性中的基质降解和周转率降低。
