Mechanisms of MMP-2 transcription in hindlimb ischemia

描述(申请人提供)：严重的肢体缺血仍然是血管外科中的一个严重问题，尽管手术搭桥和导管定向治疗，但仍有相当一部分患者最终接受截肢。在体内驱动对纠正缺血至关重要的基因转录的细胞和分子机制在很大程度上仍不清楚，这是开发组织缺血治疗方案的一个潜在领域。基质金属蛋白酶是组织缺血诱导血管生成和动脉生成的关键酶。基质金属蛋白酶2(MMP2)是一种在转录水平上诱导的组织缺血，是血管生成和动脉扩张的关键酶。基质金属蛋白酶-2转录诱导的分子机制以及在体内缺血时基质金属蛋白酶-2上调的意义尚不清楚。因此，我们建议验证这一建议的中心假设：组织缺血通过特定的顺式和反式调节元件诱导基质金属蛋白酶-2的转录，而基质金属蛋白酶-2的表达在缺血诱导的血管生成和动脉生成中起关键作用。这一假说将以以下特定目的进行检验：1)确定在缺血诱导的体内转录激活中起关键作用的基质金属蛋白酶-2启动子区域；2)确定与缺血诱导的体内基质金属蛋白酶-2转录有关的转录因子，并确定它们在体内被特定介质激活的特征；3)确定基质金属蛋白酶-2是否在缺血后的血管重建反应中起关键作用，并检测细胞类型特异性的基质金属蛋白酶-2过表达在体内缺血诱导的血管生成和动脉生成中的作用。明确在体内诱导基质金属蛋白酶-2表达的细胞和分子机制将增加我们对组织缺血引起的关键基因转录激活的了解，并为开展严重肢体缺血的分子治疗奠定基础。

DESCRIPTION (provided by applicant): Critical ischemia of the lower limbs remains a serious problem in vascular surgery with a substantial fraction of patients undergoing eventual amputation despite surgical bypass and catheter-directed therapies. The cellular and molecular mechanisms that drive in vivo transcription of genes essential for correction of ischemia remain largely undefined, and are a potential area for the development of therapeutic regimens for tissue ischemia. Matrix metalloproteinases are enzymes critical for angiogenesis and arteriogenesis induced by tissue ischemia. Matrix metalloproteinase 2 (MMP-2) is transcriptionally induced in tissue ischemia and is a critical enzyme for angiogenesis and arterial enlargement. The molecular mechanisms for transcriptional induction of MMP-2 and the significance of MMP-2 upregulation in ischemia in vivo remain unknown. Therefore, we propose to test the central hypotheses of this proposal: Tissue ischemia induces MMP-2 transcription via specific cis- and trans-acting regulatory elements and MMP-2 expression plays a critical role in the angiogenesis and arteriogenesis induced by ischemia. This hypothesis will be tested with the following Specific Aims: 1) To determine the regions within the MMP-2 promoter that are critical for in vivo transcriptional activation by ischemia, 2) To identify the transcription factors responsible for ischemia-induced MMP-2 transcription in vivo and characterize their activation by specific mediators in vivo, 3) To determine if MMP-2 is critical to the revascularization response after ischemia and to examine the effect of cell type-specific overexpression of MMP-2 on ischemia-induced angiogenesis and arteriogenesis in vivo. Defining the cellular and molecular mechanisms that induce MMP-2 expression in vivo will increase our knowledge of transcriptional activation of critical genes by tissue ischemia, and form the foundation for the development of molecular therapy for critical limb ischemia in patients.