The role of microRNAs during arteriogenesis

流体切应力(FSS)作用于血管内皮细胞，引起微核糖核酸(miRNA，miRs)表达谱改变，进而改变血流状态下血管内皮细胞的形态和功能，将原有的侧枝小动脉重塑为功能完整的传导动脉。MIR是一种小的非结合RNA，它通过抑制翻译或由于RNA衰退而在转录后调节基因的表达。在一项初步研究中，我们通过基因芯片筛选、定量RT-PCR和原位杂交鉴定了miR-24、miR-143、miR-146a和miR-195在生长侧支动脉中的差异表达。通过本项目，我们将在体外和小鼠模型中评估这些miR与动脉形成的相关性。在初步实验中，在股动脉结扎后，通过局部动脉内注射AntimiRs(单站互补RNA寡核苷酸)来阻断这些MIR，结果表明，接受AntimiR-143治疗的小鼠的血流恢复显著降低。此外，它们的后肢坏死和自体截肢增加，这导致了更高的致命性。在阐明应用抗miR-143的严重后果的原因时，将考虑mir-143作为关键调节因子的可能性以及治疗性miRNA调节的不良副作用的存在。本研究将阐明侧枝生长过程中的特异性抗iR效应(阻断靶基因)和非特异性全身抗iR效应之间的区别。一方面，将评估miRNAs的调节和分子相互作用及其在动脉形成关键过程中的作用，另一方面，将检查抗iR应用对血管止血的可能影响。已有研究表明，核糖核酸酶不是惰性结构；它们表现出毒性、促凋亡、促凝血或促炎活性。如果这也适用于反专利申请，将对其进行详细分析。

The fluid shear stress (FSS)-induced remodeling of pre-existing collateral arterioles into fully functional conductance arteries is termed "arteriogenesis".It has been shown that FFS, which is exerted on endothelial cells, leads to changes in the expression profile of microRNA (miRs), which in turn alters morphology as well as function of endothelial cells under flow conditions. miRs are small non-conding RNAs, which regulate gene expression post-transcriptionally either through inhibition of translation or due to RNA decay. In a pilot study we identified a differential expression of miR-24, miR-143, miR-146a and miR-195 in growing collateral arteries by microarray screening, quantitative RT-PCR, and in situ hybridization.Through the current project the relevance of these miRs for arteriogenesis will be evaluated in vitro and in a mouse model. In preliminary experiments, blockage of those miRs by local intra-arterial injection of antimiRs (single-standed complementary RNA oligonucleotides) after ligation of the femoral artery showed that mice, which were treated with antimiR-143, have a significantly reduced perfusion recovery. In addition they suffer from increased necrosis and autoamputation of hind limbs, which results in a higher lethality. While elucidating the cause of the drastic consequences of antimiR-143 application, the possibility of mir-143 being a key regulator and the presence of unwanted side effects of therapeutic miRNA modulation will be accounted for. The present study will clarify the differentiation between specific antimiR effects (blockage of target mRNA) during collateral growth and unspecific systemic antimiR effects. On one hand, the regulation and molecular interaction of miRNAs and their contribution to arteriogenic key processes will be assessed, on the other hand, possible influence of antimiR application on vascular hemostasis will be examined. It has been shown that ribonucleases are not inert structures; they rather display toxic, apoptotic pro-coagulatory or pro-inflammatory activity. It will be analyzed in detail if this is also true for antimiRs.