MMP REGULATION BY DOXYCYCLINE IN AORTIC ANEURYSM

腹主动脉瘤（AAA）是一种常见的致命性疾病，发病率呈上升趋势。虽然用超声波检测动脉瘤既简单又便宜，但大多数动脉瘤在检测时都很小，而且目前还没有能够抑制它们生长的药物。越来越多的证据表明，AAA组织中存在基质降解酶家族，即基质金属蛋白酶（MMPs）。尽管MMP-9和MMP-12都可能在AAA中发挥作用，但我们已经发现，AAA组织中MMP-2总量显著增加。重要的是，AAA组织中更大比例的MMP-2处于活性形式，直接与基质结合，表明正在进行蛋白水解。此外，我们已经证明AAA组织含有更高水平的膜型1 MMP， MMP-2的激活剂。我们还发现强力霉素可以抑制培养的主动脉平滑肌细胞产生MMP-2。我们假设MMP-2，通过其增加的激活，在动脉瘤形成中起着核心作用，这可以被强力霉素抑制。这一假设将通过以下具体目标进行检验：1。确定与AAA相关的单个MMPs，包括MMP-2、MT1-MMP、MMP-9和MMP-12对小鼠AAA模型中动脉瘤形成的大小和速率的影响。2. 在小鼠模型中确定强力霉素对动脉瘤形成和发展的大小和速率的影响，并将这些影响与血清强力霉素水平联系起来。3. 确定强力霉素下调人主动脉平滑肌细胞MMPs的机制。具体目标1将使用我们实验室鉴定的AAA小鼠模型来完成，该模型采用四种不同的敲除小鼠，包括MMP-2、MMP-9、MMP-12和不发生MMP-2激活的TIMP-2敲除小鼠。具体目标2将通过在我们的小鼠AAA模型中使用强力霉素治疗，以及对主动脉MMP表达、动脉瘤大小和生长速度与血清强力霉素浓度的相关性影响来实现。具体目标3将通过测定MMP-2 mRNA水平、mRNA半衰期、mRNA转录率和鉴定MMP-2启动子中的多西环素应答元件来完成。这项工作的长期目标是开发专门针对在动脉瘤发病和进展中重要的MMPs的药物治疗。

Abdominal Aortic Aneurysm (AAA) is a common and devastating disease which is increasing in incidence. Although easy and inexpensive to detect by ultrasound, most aneurysms are small when detected and there is currently no medical regimen which will inhibit their growth. There is an increasing body of evidence implicating a family of matrix degrading enzymes, the matrix metalloproteinases (MMPs) in AAA. Although both MMP-9 and MMP-12 may have a role in AAA, we have identified a significant increase in total MMP-2 in AAA. Importantly, a larger proportion of the MMP-2 in AAA tissue is in the active form and is directly bound to the matrix suggesting ongoing proteolysis. In addition, we have demonstrated that AAA tissue contains increased levels of membrane type 1 MMP, the activator of MMP-2. We have also shown that doxycycline inhibits MMP-2 production by aortic smooth muscle cells in culture. We hypothesize that MMP-2, through its increased activation, has a central role in aneurysm formation and that this could be inhibited by doxycycline. This hypothesis will be examined through the following specific aims: 1. Determine the effects of individual MMPs implicated in AAA including MMP-2, MT1-MMP, MMP-9 and MMP-12 on the size and rate of aneurysm formation in a murine AAA model. 2. Determine the effects of doxycycline on the size and rate of aneurysm formation and progression in a murine model and correlate these effects with serum doxycycline levels. 3. Determine the mechanisms by which doxycycline down regulates MMPs in human aortic smooth muscle cells. Specific aim 1 will be accomplished using a mouse model of AAA characterized in our laboratory with four different knock-out mice, including MMP-2, MMP-9, MMP-12 and a TIMP-2 knock-out mouse in which activation of MMP-2 does not occur. Specific aim 2 will be accomplished by using doxycycline treatment in our murine model of AAA and correlating effects on aortic MMP expression, aneurysm size and growth rate with serum doxycycline concentrations. Specific aim 3 will be accomplished by determining MMP- 2 mRNA levels, mRNA half life, rate of mRNA transcription and identifying the doxycycline responsive elements in the MMP-2 promotor. The long term goal of this work is to develop pharmacologic therapies which specifically target MMPs important in aneurysm pathogenesis and progression.