Rationale: Effective neovascularization is crucial for recovery after cardiovascular events. Objective: Because microRNAs regulate expression of up to several hundred target genes, we set out to identify microRNAs that target genes in all pathways of the multifactorial neovascularization process. Using www.targetscan. org, we performed a reverse target prediction analysis on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster. Microarray analyses showed upregulation of 14q32 microRNAs during neovascularization in mice after single femoral artery ligation. Methods and Results:Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4 GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased collateral artery diameters (arteriogenesis) were observed in adductor muscles of GSO-treated mice, as well as increased capillary densities (angiogenesis) in the ischemic soleus muscle. In vitro, treatment with GSOs led to increased sprout formation and increased arterial endothelial cell proliferation, as well as to increased arterial myofibroblast proliferation. Conclusions Welten et al 14q32 MicroRNAs in Neovascularization 697Both arteriogenesis and angiogenesis are highly multifactorial processes, and yet clinical trials aiming to induce neovascularization in patients with occlusive arterial disease have so far only focused on single-factor therapeutics, such as growth factors (eg, vascular endothelial growth factor A [VEGFA] and basic fibroblast growth factor [bFGF]). Unfortunately, these trials were less successful than anticipated.1,3,4 Growth factors only target 1 of multiple processes required for efficient neovascularization. Therefore, there is a need for novel proarteriogenic and proangiogenic factors that can act as master switches in neovascularization.MicroRNAs are endogenous RNA molecules that downregulate expression of their target genes.5 MicroRNAs do not completely silence their target genes, but rather downtune their expression. However, because each microRNA has multiple, up to several hundred, target genes, changes in microR-NA expression can have a major impact. Inhibition of a single microRNA can thus lead to activation of entire multifactorial physiological processes.Several studies have been published on the effects of microRNA inhibition on neovascularization, but in general, the focus of these studies lies with angiogenesis alone, not arteriogenesis. [6][7][8][9][10][11][12][13][14] In the present study, we exploited the master switch character of microRNAs to identify microRNAs that play a regulat...

R NA-binding proteins are central regulators of gene expression in both health and disease. 1,2 The RNA-binding protein Quaking (QKI) is a member of the highly conserved signal transduction and activator of RNA (STAR) family of RNA-binding proteins. 3 Alternative splicing of the mammalian qkI transcript yields 3 protein isoforms, notably QKI-5, QKI-6, and QKI-7, 2 with dimerization of QKI isoforms being required for the regulation of pre-mRNA splicing, mRNA export, and stability. 2,4 QKI drives central and peripheral nervous system myelination by regulating oligodendrocyte and Schwann cell differentiation, respectively. 2,4,5 However, a role for QKI outside the neural network is poorly understood. In This

IntroductionAngiogenesis in the adult is associated with specific conditions such as tissue ischemia and wound repair, in which the formation of new blood vessels is temporally and spatially controlled. Tissue injury causes damage of blood vessels and the extravasation of plasma proteins, including fibrinogen, which results in the formation of a fibrin clot in the surrounding interstitium. This extracellular matrix of extravasated fibrin entangled with the existing collagen fibers is furthermore composed of a number of proteins such as vitronectin, fibronectin, laminin, hyaluronic acid, and proteoglycans. The fibrinous matrix formed serves as a provisional matrix, into which cells can infiltrate during the subsequent wound healing. [1][2][3] During the formation of a granulation tissue, a dynamic interaction between microvascular endothelial cells (MVECs) and the surrounding extracellular matrix occurs. The cells disrupt existing cell-matrix interactions and locally degrade the surrounding extracellular matrix; they migrate, proliferate, and form new capillarylike tubular structures, which become stabilized in the course of time. 4,5 The temporary fibrin matrix can be degraded by plasmin, which is activated from its zymogen by 2 types of plasminogen activators, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). 6 The activity of u-PA is directed to the cell surface by a cellular u-PA receptor (u-PAR). 7 In vitro 8,9 and in vivo 10,11 studies have shown that the u-PA/plasmin system plays a critical role in the process of angiogenesis. In addition to the u-PA/plasmin system, matrix metalloproteinases (MMPs) are also involved in the degradation of the extracellular matrix. [10][11][12][13][14][15] A role for both u-PA/plasmin and MMP systems in angiogenesis is supported by the fact that different components in the extracellular matrix are substrates for plasmin as well as for MMPs, including fibrin, 16 vitronectin, fibronectin, laminin, gelatins, and proteoglycans, whereas plasmin is unable to degrade collagens. 17 Furthermore, endothelial cells at the leading edge of a new blood vessel concomitantly express components of both protease systems, [18][19][20] and their expression is regulated by the same growth factors and cytokines. [21][22][23][24] MMPs are a family of zinc-dependent enzymes that can be divided into 2 structurally distinct groups, the secreted MMPs and the membrane-type MMPs (MT-MMPs). The MMPs are secreted as inactive zymogens, and in vitro studies have shown that the u-PA/plasmin system located at the cell membrane directly or indirectly activates a number of pro-MMPs, such as pro-MMP-1, pro-MMP-3, pro-MMP-9, pro-MMP-10, and pro-MMP-13. 25,26 In addition to zymogen activation, MMP activity is regulated by tissue inhibitors of metalloproteinases (TIMPs), of which to date 4 types have been characterized. 27 MT-MMPs, of which 6 types are known, are transmembrane proteins that are activated intracellularly in the secretory pathway by furinlike enzymes. 28-30 MT1...