Department of Genetics, Boys Town National Research Hospital, Omaha, Nebraska 68131, USA.
Curr Eye Res. 2010 Jan;35(1):45-55. doi: 10.3109/02713680903374208.
The potential role of arresten (alpha1(IV)NC1) as an endogenous angiogenesis inhibitor in the prevention of bFGF mediated retinal angiogenesis and regulation of matrix metalloproteinase-2 activation has not been explored.
Mouse retinal endothelial cells (MREC) were cultured on type IV collagen and treated with basic fibroblast growth factor (bFGF) alone or in the presence of arresten at concentrations ranging from 1 to 10 microg/ml. The proliferation of MRECs were evaluated using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, and bFGF stimulated endothelial cell migration was assessed using Boyden chamber. Expression of matrix metalloproteinase-2 (MMP-2) was assessed by reverse transcription polymerase chain reaction (RT-PCR) analysis using RNA isolated from MRECs. Secretion and activation of MMP-2 in arresten-treated conditioned MREC growth medium was determined by gelatin zymography and Western blotting.
Different doses of bFGF induced MREC proliferation was significantly inhibited upon arresten treatment (P < 0.005). The bFGF-induced migration was significantly inhibited by arresten at 1 and 10 microg/ml concentrations (P < 0.01). The bFGF stimulated expression of MMP-2 mRNA and secretion of MMP-2 in MREC was not affected and interestingly activation of MMP-2 was suppressed by arresten in a dose and time dependent manner.
Inhibitory effects of arresten on proliferation, migration and MMP-2 activation but not on expression and secretion of MMP-2 in MREC; this early work with arresten supports potential therapeutic action in retinal neovascularization dependent disorders.
尚未探索 arresten（alpha1(IV)NC1）作为内源性血管生成抑制剂在预防 bFGF 介导的视网膜血管生成和调节基质金属蛋白酶-2 激活中的潜在作用。
在 IV 型胶原上培养小鼠视网膜内皮细胞（MREC），并用碱性成纤维细胞生长因子（bFGF）单独或在浓度为 1 至 10 微克/毫升的 arresten 存在下处理。使用 3（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐（MTT）比色法评估 MRECs 的增殖，并用 Boyden 室评估 bFGF 刺激的内皮细胞迁移。使用从 MRECs 分离的 RNA 通过逆转录聚合酶链反应（RT-PCR）分析评估基质金属蛋白酶-2（MMP-2）的表达。通过明胶酶谱法和 Western blot 测定 arresten 处理的条件 MREC 生长培养基中 MMP-2 的分泌和激活。
不同剂量的 bFGF 诱导的 MREC 增殖在 arresten 处理后显著受到抑制（P <0.005）。在 1 和 10 微克/毫升浓度下，arresten 显著抑制 bFGF 诱导的迁移（P <0.01）。bFGF 刺激的 MMP-2 mRNA 表达和 MREC 中 MMP-2 的分泌不受影响，有趣的是，MMP-2 的激活被 arresten 以剂量和时间依赖的方式抑制。
arresten 对增殖、迁移和 MMP-2 激活的抑制作用，但对 MMP-2 在 MREC 中的表达和分泌没有影响；这项关于 arresten 的早期工作支持其在依赖视网膜新生血管形成的疾病中的潜在治疗作用。
