P.H.A. Quax

Inhibition of Individual 14q32 MicroRNAs Drastically Increases Neovascularization and Blood Flow Recovery after Ischemia

Introduction and Hypothesis: Neovascularization, i.e. angiogenesis and arteriogenesis, is a multifactorial process. As microRNAs can regulate expression of up to several hundred target genes, we hypothesized that specific microRNAs may target not just single aspects of neovascularization, but neovascularization as a whole. We set out to identify microRNAs that target genes in all pathways of neovascularization. Using www.targetscan.org, we performed a reverse target prediction on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster on the long arm of human chromosome 14. MicroArray analyses showed that 14q32 microRNAs were down-regulated during effective neovascularization in mice subjected to single femoral artery ligation. Methods and Results: Gene Silencing Oligonucleotides (GSOs), were injected (1mg/mouse) to inhibit four 14q32 microRNAs, miR-487b, miR-494, miR-329 and miR-495, one day prior to double ligation of the femoral artery. 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 vs. 15% in control animals) and those treated with GSO-329 showed a remarkable full recovery of perfusion after 7 days (vs. 60% perfusion in control animals). In vivo arteriogenesis was enhanced as 3-fold increased collateral artery diameters were observed in adductor muscles of GSO-treated mice. Simultaneously, in vivo angiogenesis was also enhanced as we observed up to 10-fold increased capillary densities in the ischemic soleus muscles of GSO-treated mice. Furthermore, in vitro treatment with GSO-329, GSO-495 and GSO-487b led to increased proliferation of primary human arterial endothelial cells whereas treatment with GSO-494 led to increased proliferation of primary human arterial fibroblasts. Conclusions: Inhibition of 14q32 microRNAs leads to drastic increases in post-ischemic blood flow recovery in vivo via stimulation of both arteriogenesis and angiogenesis. In conclusion, 14q32 microRNA inhibition may offer an alternative to growth factors in therapeutic neovascularization.