Mechanisms of MMP-2 transcription in hindlimb ischemia

描述（由申请人提供）：下肢严重缺血在血管手术中仍然是一个严重的问题，尽管手术搭桥和导管指导治疗，仍有相当一部分患者最终截肢。驱动体内基因转录的细胞和分子机制在很大程度上仍然不明确，这是组织缺血治疗方案发展的潜在领域。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 （MMP-2）在组织缺血时被转录诱导，是血管生成和动脉扩张的关键酶。体内MMP-2转录诱导的分子机制以及MMP-2上调在缺血中的意义尚不清楚。因此，我们提出验证该提议的中心假设：组织缺血通过特定的顺式和反式调控元件诱导MMP-2转录，MMP-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。这一假设将通过以下具体目的进行检验：1)确定MMP-2启动子内对缺血在体内的转录激活至关重要的区域；2)确定负责缺血诱导的MMP-2转录的转录因子，并表征它们在体内被特定介质激活的特征。3)确定MMP-2是否在缺血后血运重建反应中起关键作用，并在体内检测细胞类型特异性过表达MMP-2对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，并为发展重症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.