微小RNA miR-34a导致老年血管新生障碍的机制研究

Impaired vascular repair/angiogenesis is a major clinical problem in aged patients that often leads to ischemic peripheral artery and cardiovascular diseases. Recent studies including ours suggest that endothelial progenitor cells (EPCs), precursor of mature endothelial cells, play an important role in angiogenesis, which could be used for autologous cell therapy of various vascular diseases. However, EPC angiogenic functions are severely impaired in aging, directly limiting their successful use clinically. MicroRNAs (miRNAs) are a new class of small endogenous non-coding RNAs that may exert their therapeutic potential as key post-transcriptional regulators. Only a few miRNAs have been reported to exert pro- or anti-angiogenic effects in mature endothelial cells to date. Remarkably, little information exists on miRNA regulation of EPC-mediated angiogenesis, especially under aging. This proposal focuses on microRNA mir-34a because our preliminary studies indicate that it is significantly up-regulated in aged EPCs, and its suppression rescues EPC functions and senescence in aging. The mechanisms underlying this fascinating phenomenon remain unclear. We hypothesize that miR-34a retards EPC angiogenesis in aging by directly inhibiting GTP-Cyclohydrolase 1 (GCH1)/Tetrahydrobiopterin (BH4) pathway, resulting in excessive oxidative stress, decreased telomerase activity and accelerated senescence. This hypothesis was formulated after a careful analysis of published work in the field and the generation of some key preliminary data in our laboratory. Firstly, we will determine how miR-34a inhibits EPC-mediated angiogenesis in aging. Our working hypothesis is that one mechanism underlying impaired EPC angiogenesis by miR-34a in aging via blockades of GCH1/BH4 pathway. Secondly, we will determine how GCH1/BH4 deficiency contributes to miR-34a-induced impairment of EPC angiogenesis. We hypothesize that BH4 reduction augments EPC oxidative stress, leading to decreased telomerase activity and increased senescence. Finally, we will determine for the first time the functional consequence of targeting miR-34a for EPC cell therapy on angiogenesis in aging. We hypothesize that suppression of miR-34a in aged EPCs results in enhanced angiogenesis in aged mice. The major significance of this study is that it will determine how miRNAs regulate EPC angiogenic functions during aging. Once such knowledge is gained, it is possible that new modalities may be developed to therapeutically rescue EPC dysfunction in the growing number of aging patients today.

机体老化会伴有血管自我修复能力及血管新生功能降低，容易诱发血管损伤性疾病。当缺血或内皮受损时，内皮祖细胞（EPCs）可以归巢到缺血部位并分化为成熟内皮细胞，从而参与缺血区域的血管新生。然而，老年患者尤其有心血管基础疾病的老年患者的EPC的数量及功能都明显降低，并直接影响EPC的治疗效果。我们假设微小RNA分子 miR-34a通过抑制GCH I/BH4 通路，促进EPC细胞内氧自由基生成，降低端粒酶活性，并加速衰老发生，最后导致EPC/内皮功能障碍和受损血管修复能力降低。 本研究旨在从微小RNA及EPCs的角度研究老年化相关的血管损伤机制，并探索可能的治疗策略。我们希望通过本项目的研究，能够阐明miRNA 调节内源性GCH 1/BH4的的机制，为血管损伤性疾病的微小RNA基因治疗或EPCs细胞治疗提供理论依据与实验支持。

机体的老化会伴有血管内皮的自我修复能力及血管新生功能降低，容易诱发血管损伤性疾病。当缺血或内皮受损时，内皮祖细胞（EPCs）可以归巢到缺血部位并分化为成熟内皮细胞，从而参与缺血区域的血管新生。然而，老年患者尤其有心血管基础疾病的老年患者的EPC的数量及功能都明显降低，并直接影响EPC的治疗效果。我们结果显示当用miR-34a抑制剂转染老年小鼠EPCs 72小时后，EPC内的miR-34水平被抑制了约90%，并且老年小鼠EPC的小管成形能力，粘附功能比为转染小鼠EPC的功能有明显的改善。相反的，我们将miR-34a类似物转染老年小鼠EPCs 72小时后，年轻小鼠EPC的小管成形能力和粘附功能都较对照组明显增多. 实验结果表明外源性高表达miR-34a后EPC的小管成形能力、衰老数量较年轻老鼠没有统计学差异，与老龄野生型小鼠的EPC的小管成形能和衰老仍有统计学差异。 并且经miR-34a类似物处理后的年轻小鼠EPC 内的氧自由基水平比对照未处理组明显增高（1.5~1.8倍）； 而经miR-34a抑制剂处理后的老年小鼠EPC 的氧自由基水平则比对照组受到30~50% 抑制。 与此同时，实验结果表明当与年轻小鼠EPC比较时，端粒酶的活性在年老小鼠EPC内是明显降低的； 而在miR-34a类似物处理后，年轻小鼠EPC 的端粒酶活性被抑制；与此对应，老年小鼠EPC经miR-34a的抑制剂处理后其端粒酶活性得到了保护。当与Tg-GCH小鼠EPC比较时，端粒酶的活性在hph-1小鼠EPC内是明显降低的；而在miR-34a类似物处理后，Tg-GCH小鼠EPC 的端粒酶活性被抑制；相反的，hph-1小鼠EPC经miR-34a的抑制剂处理后其端粒酶活性得到了保护。在细胞衰老试验中，发现miR-34a抑制剂可以hph-1老年小鼠EPC的衰老，而miR-34a的类似物促进Tg-GCH小鼠EPC 的衰老；实验结果显示miR-34a可能通过调节GCH/BH4通路增加细胞内氧自由基，抑制端粒酶的活性，从而促进细胞的衰老。老年小鼠EPC内的eNOS 水平较年轻小鼠EPC降低并进一步比较在miR-34a类似物或抑制剂处理后对EPCs内NO的活性及eNOS水平的影响。而eNOS基因敲除小鼠（eNOS KO小鼠）EPC内miR-34a的水平较野生型小鼠高，同时EPC的小管成行能力，粘附功能都较低。本项目的