(C) Phosophorylated ERK1/2 expression in the thigh WT and KO mice on 14?days after hindlimb ischaemia as determined by Western blot on day 14. of great interest for ischaemic diseases, little is known about the modulation of the signalling cascades microRNAs. We observed that miR-132/212 expression was significantly upregulated after occlusion of the femoral artery. miR-132/212 knockout (KO) mice display a slower perfusion recovery after hind-limb ischaemia compared to wildtype (WT) mice. Immunohistochemical analysis demonstrates a clear trend towards smaller collateral arteries in KO mice. Although aortic ring assays score similar number of branches in miR-132/212 KO mice compared to WT, it can be stimulated with MAPT exogenous miR-132, a dominant member of the miR-132/212 family. Moreover, in pericyte-endothelial co-culture cell assays, overexpression of miR-132 and mir-212 in endothelial cells results in enhanced vascularization, as shown by an increase in tubular structures and junctions. Our results suggested that miR-132/212 may exert their effects by enhancing the Ras-Mitogen-activated protein kinases MAPK signalling pathway through direct inhibition of Rasa1, and Spred1. The miR-132/212 cluster promotes arteriogenesis by modulating Ras-MAPK signalling direct targeting of its inhibitors Rasa1 and Spred1. arteriogenesis weights much more than the number of newly formed capillaries angiogenesis and has therefore the potential to become a future therapeutic approach 4 in chronic and acute ischaemic diseases. Many attempts have been made to modulate the pro- and anti-arteriogenic balance 5C7. However, effective therapeutic approaches to promote arteriogenesis are still lacking. Initial studies have shown an important role for microRNAs (miRNAs) in neovascularization 8C14, but a clear understanding of all players involved is still lacking. It has previously been shown that miR-132 is upregulated in endothelial cells by various pro-angiogenic stimuli such as hypoxia 15, VEGF 10,15, and angiotensin II 16. Overexpression of miR-132 in human umbilical venous endothelial cells (HUVECs) promoted proliferation and migration and transplanting these cells promoted vascularization assays and animal models to explore the role of miR-132/212 in vascular growth during arteriogenesis and to unravel the underlying mechanism. Materials and methods Generation and genotyping of miR-132/212 KO mice The generation of miR-132/212 KO mice has been described as previously 20. For genotyping, DNA samples were obtained by ear clipping and used in a GC-Rich PCR kit (Cat. 12140306001; Roche, Switzerland) with the MiR-132/212 primers as shown in the Table?S1. PCR products were revealed on a 1% agarose gel: wildtype (WT) genotype shows a predicted band at 1076?bp and the KO genotype at 392?bp. Hind-limb ischaemia This study was approved by the Animal Ethical Experimentation Committee (Utrecht University) and was carried out in accordance with the Guide for the care and use of Laboratory Animals. Hind-limb ischaemia was applied on 10C12?week old mice [10 WT (C57B6) and 13 miR-132/212 KO] as described previously 21. In brief, mice were anaesthetized with fentanyl (0.05?mg/kg), midazolam (5?mg/kg) and medetomidine (0.5?mg/kg) by intraperitoneal injection and surgical procedures were performed under sterile conditions. A vertical Dehydrocostus Lactone longitudinal incision was made in the right hind-limb and the femoral artery was dissected. To achieve slower recovery, ligation was performed using an electricoagulator at Dehydrocostus Lactone the most proximal position and thereby separating them into two parts. After closure, mice received atipamezole (2.5?mg/kg) and flumazenil (0.5?mg/kg) to recover. Temgesic (0.1?mg/kg) was given every Dehydrocostus Lactone 8?hrs after surgery for 6 times. Measurement of blood flow was performed by scanning both rear paws with an LDI analyzer (Moor Infrared Laser Doppler Imager Instrument, Wilmington, DE, USA), before and after the surgical procedure (days 0, 4, 7, and 14). During the procedure, the animal was kept under 2% isoflurane anaesthesia and its body temperature was strictly maintained between 36.5 and 37.5C. The images obtained were quantitatively converted into histograms with Moor LDI processing software as described before 22. Data were reported as the ratio of blood flow in the right over left (R/L) hindlimb. MicroRNA hybridization The procedure for microRNA hybridization has been described previously with slight modification 23. Cryosections were fixed by 4% paraformaldehyde for 10?min., acetylated for 10?min. followed with 10?min. proteinase K treatment (10?g/ml). Hybridization Dehydrocostus Lactone was performed following manufacturers suggestions with DIG labelled miRCURY LNA miRNA detection probes (Exiqon, Vedbaek, Denmark) for miR-132 (38031-15), negative control miR-159 (99003-15) and positive control U6 (99002-15). Sections were subsequently blocked for 1?hr before overnight incubation with anti-DIG alkaline phosphatase antibody (1:1500; Roche, Switzerland). To block endogenous alkaline phosphatase activity, sections were incubated with levamisole solution (DAKO, USA), followed by Liquid Permanent Red (DAKO, USA) incubation for visualization. Blood vessels were stained with lectin BS-1 (1:100; Sigma-Aldrich, USA). Nuclei were stained with Hoechst 33342 (Life Technologies, USA). Images were taken by Zeiss LSM710 and analysed using Zen2012 (Zeiss, Germany). RNA isolation and RT-PCR DNA-free RNA was extracted with Tripure (Roche Applied Science, Switzerland). To perform quantitative PCR.