Mechanisms of MMP-2 transcription in hindlimb ischemia

描述（由申请人提供）：下肢严重缺血仍然是血管外科中的一个严重问题，尽管有外科搭桥和导管导向治疗，但仍有相当一部分患者最终接受截肢。驱动对纠正缺血必不可少的基因的体内转录的细胞和分子机制在很大程度上仍然不确定，并且是开发组织缺血治疗方案的潜在领域。基质金属蛋白酶是由组织缺血诱导的血管生成和动脉形成的关键酶。基质金属蛋白酶2（MMP-2）在组织缺血中被转录诱导，是血管生成和动脉扩张的关键酶。MMP-2的转录诱导的分子机制和MMP-2上调在体内缺血中的意义仍然未知。因此，我们建议测试这个建议的中心假设：组织缺血诱导MMP-2的转录通过特定的顺式和反式作用的调节元件和MMP-2的表达在缺血诱导的血管生成和动脉生成中起着至关重要的作用。该假设将通过以下具体目标进行检验：1）确定MMP-2启动子内对缺血引起的体内转录激活至关重要的区域，2）鉴定负责缺血诱导的MMP-2体内转录的转录因子，并表征它们在体内被特异性介体激活的特征，3）确定MMP-2是否是缺血后血管再生反应的关键，并检查MMP-2的细胞类型特异性过表达对体内缺血诱导的血管生成和动脉生成的影响。明确体内诱导MMP-2表达的细胞和分子机制将增加我们对组织缺血关键基因转录激活的认识，并为发展严重肢体缺血患者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.