miR-96 and miR-183 differentially regulate neonatal and adult postinfarct neovascularization
Raphael F.P. Castellan, Milena Vitiello, Martina Vidmar, Steven Johnstone, Dominga Iacobazzi, David Mellis, Benjamin Cathcart, Adrian Thomson, Christiana Ruhrberg, Massimo Caputo, David E. Newby, Gillian A. Gray, Andrew H. Baker, Andrea Caporali, Marco Meloni
Raphael F.P. Castellan, Milena Vitiello, Martina Vidmar, Steven Johnstone, Dominga Iacobazzi, David Mellis, Benjamin Cathcart, Adrian Thomson, Christiana Ruhrberg, Massimo Caputo, David E. Newby, Gillian A. Gray, Andrew H. Baker, Andrea Caporali, Marco Meloni
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Research Article Angiogenesis Vascular biology

miR-96 and miR-183 differentially regulate neonatal and adult postinfarct neovascularization

Abstract

Following myocardial infarction (MI), the adult heart has minimal regenerative potential. Conversely, the neonatal heart can undergo extensive regeneration, and neovascularization capacity was hypothesized to contribute to this difference. Here, we demonstrate the higher angiogenic potential of neonatal compared with adult mouse cardiac endothelial cells (MCECs) in vitro and use this difference to identify candidate microRNAs (miRs) regulating cardiac angiogenesis after MI. miR expression profiling revealed miR-96 and miR-183 upregulation in adult compared with neonatal MCECs. Their overexpression decreased the angiogenic potential of neonatal MCECs in vitro and prevented scar resolution and neovascularization in neonatal mice after MI. Inversely, their inhibition improved the angiogenic potential of adult MCECs, and miR-96/miR-183–KO mice had increased peri-infarct neovascularization. In silico analyses identified anillin (ANLN) as a direct target of miR-96 and miR-183. In agreement, Anln expression declined following their overexpression and increased after their inhibition in vitro. Moreover, ANLN expression inversely correlated with miR-96 expression and age in cardiac ECs of cardiovascular patients. In vivo, ANLN+ vessels were enriched in the peri-infarct area of miR-96/miR-183–KO mice. These findings identify miR-96 and miR-183 as regulators of neovascularization following MI and miR-regulated genes, such as anillin, as potential therapeutic targets for cardiovascular disease.

Authors

Raphael F.P. Castellan, Milena Vitiello, Martina Vidmar, Steven Johnstone, Dominga Iacobazzi, David Mellis, Benjamin Cathcart, Adrian Thomson, Christiana Ruhrberg, Massimo Caputo, David E. Newby, Gillian A. Gray, Andrew H. Baker, Andrea Caporali, Marco Meloni

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Figure 6

Inhibition of miR-96 and miR-183 increase neovascularization in the adult mouse after MI.

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Inhibition of miR-96 and miR-183 increase neovascularization in the adul...
(A) Bar graph showing the increased number of capillaries (n = 5/group) and (B) small arterioles (n = 6 WT, n = 7 miR-96/miR-183 KO) in the peri-infarct of miR-96/miR-183–KO mice at 14 days after MI. *P < 0.05 vs. WT (Student’s t test). (C) Representative microphotographs showing capillaries (stained by isolectin-B4, green fluorescence) and arterioles (stained by isolectin-B4, green fluorescence, and α-smooth muscle actin, red fluorescence). Scale bar: 250 μm (left); 50 μm (right). (D) Bar graph and representative microphotographs showing the increased percentage of ANLN+ vessels in the peri-infarct of miR-96/miR-183–KO mice at 14 days after MI (n = 6/group). ECs are stained by isolectin-B4 (green fluorescence). ANLN, red fluorescence. Scale bar: 20 μm. *P < 0.05 vs. WT (Student’s t test). (E) Adult mouse cardiac endothelial cells (MCECs) were transduced with lentiviral vectors LV-EGFP or LV-ANLN-EGFP and seeded on Matrigel. Representative Matrigel assay images and quantification as total tubule length (n = 5/group). Scale bar: 100 μm. **P < 0.01 vs. LV-EGFP (Student’s t test). Data are shown as mean ± SEM.