MMP REGULATION BY DOXYCYCLINE IN AORTIC ANEURYSM

腹主动脉瘤(AAA)是一种常见病、危害性疾病，其发病率呈上升趋势。虽然通过超声波检测很容易而且便宜，但大多数动脉瘤在被检测到时都很小，目前还没有抑制其生长的药物方案。越来越多的证据表明，AAA中存在一类基质降解酶，即基质金属蛋白酶(MMPs)。尽管基质金属蛋白酶-9和基质金属蛋白酶-12可能在腹主动脉瘤中起一定作用，但我们发现腹主动脉瘤中总基质金属蛋白酶-2的表达显著增加。重要的是，AAA组织中较大比例的基质金属蛋白酶-2处于活性状态，并直接与基质结合，提示正在进行蛋白质分解。此外，我们还证明了AAA组织中含有增加的膜型1-基质金属蛋白酶，它是基质金属蛋白酶-2的激活剂。我们还发现多西环素抑制培养的主动脉平滑肌细胞产生基质金属蛋白酶-2。我们推测，基质金属蛋白酶-2，通过其增加的活性，在动脉瘤的形成中起中心作用，这可以被多西环素抑制。这一假说将通过以下具体目标来验证：1.在小鼠AAA模型中，确定参与AAA的单个MMPs包括MMP2、MT1-MMP1、MMP9和MMP12对动脉瘤的大小和形成速度的影响。2.在小鼠模型中确定多西环素对动脉瘤的大小、形成和进展的影响，并将这些影响与血清多西环素水平相关联。3.确定多西环素下调人主动脉平滑肌细胞MMPs表达的机制。具体目标1将使用我们实验室以四种不同基因敲除小鼠为特征的AAA小鼠模型来实现，包括MMP2、MMP9、MMP12和TIMP-2基因敲除小鼠，在该小鼠中不发生MMP2的激活。在我们的小鼠AAA模型中，通过使用多西环素治疗可以实现特定的目标2，并将多西环素对主动脉基质金属蛋白酶表达、动脉瘤大小和生长速度的影响与血清多西环素浓度相关联。具体目标3将通过测定基质金属蛋白酶-2的mRNA水平、半衰期、转录速率和鉴定基质金属蛋白酶-2启动子中的多西环素反应元件来实现。这项工作的长期目标是开发专门针对在动脉瘤发生和发展中重要的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.