Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3′ UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo.regulatory RNA | bone biology | osteoblastic differentiation

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...

See also Ragni MV. On the cutting edge: von Willebrand factor propeptide and thrombosis. This issue, pp 2553-5.

Vascular remodelling is a multifactorial process that involves both adaptive and maladaptive changes of the vessel wall through, among others, cell proliferation and migration, but also apoptosis and necrosis of the various cell types in the vessel wall. Vascular remodelling can be beneficial, e.g. during neovascularization after ischaemia, as well as pathological, e.g. during atherosclerosis and aneurysm formation. In recent years, it has become clear that microRNAs are able to target many genes that are involved in vascular remodelling processes and either can promote or inhibit structural changes of the vessel wall. Since many different processes of vascular remodelling are regulated by similar mechanisms and factors, both positive and negative vascular remodelling can be affected by the same microRNAs. A large number of microRNAs has been linked to various aspects of vascular remodelling and indeed, several of these microRNAs regulate multiple vascular remodelling processes, including both the adaptive processes angiogenesis and arteriogenesis as well as maladaptive processes of atherosclerosis, restenosis and aneurysm formation. Here, we discuss the multifactorial role of microRNAs and microRNA clusters that were reported to play a role in multiple forms of vascular remodelling and are clearly linked to cardiovascular disease (CVD). The microRNAs reviewed are miR-126, miR-155 and the microRNA gene clusters 17-92, 23/24/27, 143/145 and 14q32. Understanding the contribution of these microRNAs to the entire spectrum of vascular remodelling processes is important, especially as these microRNAs may have great potential as therapeutic targets for treatment of various CVDs.