MMP REGULATION BY DOXYCYCLINE IN AORTIC ANEURYSM

腹主动脉瘤（AAA）是一种常见的破坏性疾病，其发病率呈上升趋势。虽然通过超声检测容易且便宜，但大多数动脉瘤在检测时都很小，并且目前没有抑制其生长的医学方案。越来越多的证据表明AAA中存在一个基质降解酶家族，即基质金属蛋白酶（MMPs）。虽然MMP-9和MMP-12可能在AAA中起作用，但我们已经确定AAA中总MMP-2显著增加。重要的是，AAA组织中较大比例的MMP-2是活性形式，并直接结合到基质上，表明正在进行蛋白水解。此外，我们已经证明，AAA组织含有增加水平的膜1型MMP，MMP-2的激活剂。我们还发现强力霉素抑制培养的主动脉平滑肌细胞产生MMP-2。我们假设MMP-2通过其增加的活化在动脉瘤形成中具有中心作用，并且这可以被多西环素抑制。将通过以下具体目标来检验这一假设：1。在小鼠AAA模型中确定与AAA有关的单个MMP（包括MMP-2、MT 1-MMP、MMP-9和MMP-12）对动脉瘤形成的大小和速率的影响。2.在小鼠模型中确定多西环素对动脉瘤形成和进展的大小和速率的影响，并将这些影响与血清多西环素水平相关联。3.确定强力霉素下调人主动脉平滑肌细胞基质金属蛋白酶的机制。具体目标1将使用在我们实验室中表征的AAA小鼠模型来实现，所述AAA小鼠模型具有四种不同的敲除小鼠，包括MMP-2、MMP-9、MMP-12和TIMP-2敲除小鼠，其中MMP-2的活化不发生。具体目标2将通过在我们的AAA小鼠模型中使用多西环素治疗并将对主动脉MMP表达、动脉瘤大小和生长速率的影响与血清多西环素浓度相关联来实现。具体目标3将通过测定MMP- 2 mRNA水平、mRNA半衰期、mRNA转录速率和鉴定MMP-2启动子中的多西环素应答元件来实现。这项工作的长期目标是开发专门针对在动脉瘤发病机制和进展中重要的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.