Curci J A, Mao D, Bohner D G, Allen B T, Rubin B G, Reilly J M, Sicard G A, Thompson R W
Department of Surgery (Section of Vascular Surgery), Washington University School of Medicine, St Louis, MO 63110, USA.
J Vasc Surg. 2000 Feb;31(2):325-42. doi: 10.1016/s0741-5214(00)90163-0.
Matrix metalloproteinases (MMPs) are considered to play a central role in the pathogenesis of abdominal aortic aneurysms (AAAs). Doxycycline (Dox) has direct MMP-inhibiting properties in vitro, and it effectively suppresses the development of elastase-induced AAAs in rodents. The purpose of this study was to determine if treatment with Dox suppresses MMPs within human aneurysm tissue and to elucidate the molecular mechanisms underlying this effect.
Aneurysm tissues were obtained from 15 patients with an AAA, eight of whom had been treated with Dox before surgery (100 mg orally twice a day for 7 days). Protein extracts were examined by means of gelatin zymography and immunoblot analysis, and RNA was examined by means of reverse transcription-polymerase chain reaction (RT-PCR). The effects of Dox on MMP production were further examined in human THP-1 mononuclear phagocytes in vitro.
No detectable difference was found between groups by using substrate zymography as a means of assessing total MMP activity, but Dox treatment was associated with a slight (24.4%) reduction in the activated fraction of 72-kDa gelatinase (MMP-2; P <.05). In contrast, a 2.5-fold reduction in the amount of extractable 92-kDa gelatinase (MMP-9) protein in Dox-treated patients was revealed by means of immunoblot analysis (P <.05). Also, a 5.5-fold (81.9%) reduction in MMP-9 messenger RNA (mRNA) in Dox-treated patients was demonstrated by means of quantitative competitive RT-PCR (mean +/- SE, mol MMP-9/mol beta-actin: 1.3 +/- 0.5 vs 7.2 +/- 3.1; P <.04). There was no significant difference between groups in the relative expression of MMP-2 protein or mRNA. In cultured THP-1 monocytes stimulated with phorbol ester, the expression of MMP-9 protein and mRNA were both decreased after exposure to relevant concentrations of Dox in vitro.
In addition to its recognized effects as a direct MMP antagonist, Dox may influence connective tissue degradation within human aneurysm tissue by reducing monocyte/macrophage expression of MMP-9 mRNA and by suppressing the post-translational processing (activation) of proMMP-2. Through this complementary combination of mechanisms, treatment with Dox may be a particularly effective strategy for achieving MMP inhibition in patients with an AAA.
基质金属蛋白酶（MMPs）被认为在腹主动脉瘤（AAAs）的发病机制中起核心作用。强力霉素（Dox）在体外具有直接抑制MMPs的特性，并且能有效抑制啮齿动物中弹性蛋白酶诱导的AAAs的发展。本研究的目的是确定Dox治疗是否能抑制人动脉瘤组织中的MMPs，并阐明这种作用的分子机制。
从15例AAA患者获取动脉瘤组织，其中8例在手术前接受了Dox治疗（口服100mg，每日2次，共7天）。通过明胶酶谱法和免疫印迹分析检测蛋白质提取物，通过逆转录-聚合酶链反应（RT-PCR）检测RNA。在体外人THP-1单核吞噬细胞中进一步研究Dox对MMP产生的影响。
使用底物酶谱法评估总MMP活性时，两组之间未发现可检测到的差异，但Dox治疗与72kDa明胶酶（MMP-2）的活化部分轻微降低（24.4%）相关（P<.05）。相比之下，免疫印迹分析显示Dox治疗患者中可提取的92kDa明胶酶（MMP-9）蛋白量减少了2.5倍（P<.05）。此外，定量竞争性RT-PCR显示Dox治疗患者中MMP-9信使核糖核酸（mRNA）减少了5.5倍（81.9%）（平均值±标准误，MMP-9/β-肌动蛋白摩尔比：1.3±0.5对7.2±3.1；P<.04）。两组之间MMP-2蛋白或mRNA的相对表达无显著差异。在用佛波酯刺激的培养THP-1单核细胞中，体外暴露于相关浓度的Dox后，MMP-9蛋白和mRNA的表达均降低。
除了其作为直接MMP拮抗剂的公认作用外，Dox可能通过降低单核细胞/巨噬细胞MMP-9 mRNA的表达以及抑制前MMP-2的翻译后加工（激活）来影响人动脉瘤组织中的结缔组织降解。通过这种机制的互补组合，Dox治疗可能是在AAA患者中实现MMP抑制的一种特别有效的策略。
