MicroRNA-26a regulates pathological and physiological angiogenesis by targeting BMP/SMAD1 signaling - PubMed (original) (raw)

. 2013 Nov 8;113(11):1231-41.

doi: 10.1161/CIRCRESAHA.113.301780. Epub 2013 Sep 18.

A K M Wara, Javid Moslehi, Xinghui Sun, Eva Plovie, Meghan Cahill, Julio F Marchini, Andrew Schissler, Robert F Padera, Jianru Shi, Hui-Wen Cheng, Srilatha Raghuram, Zoltan Arany, Ronglih Liao, Kevin Croce, Calum MacRae, Mark W Feinberg

Affiliations

MicroRNA-26a regulates pathological and physiological angiogenesis by targeting BMP/SMAD1 signaling

Basak Icli et al. Circ Res. 2013.

Abstract

Rationale: The rapid induction and orchestration of new blood vessels are critical for tissue repair in response to injury, such as myocardial infarction, and for physiological angiogenic responses, such as embryonic development and exercise.

Objective: We aimed to identify and characterize microRNAs (miR) that regulate pathological and physiological angiogenesis.

Methods and results: We show that miR-26a regulates pathological and physiological angiogenesis by targeting endothelial cell (EC) bone morphogenic protein/SMAD1 signaling in vitro and in vivo. MiR-26a expression is increased in a model of acute myocardial infarction in mice and in human subjects with acute coronary syndromes. Ectopic expression of miR-26a markedly induced EC cycle arrest and inhibited EC migration, sprouting angiogenesis, and network tube formation in matrigel, whereas blockade of miR-26a had the opposite effects. Mechanistic studies demonstrate that miR-26a inhibits the bone morphogenic protein/SMAD1 signaling pathway in ECs by binding to the SMAD1 3'-untranslated region, an effect that decreased expression of Id1 and increased p21(WAF/CIP) and p27. In zebrafish, miR-26a overexpression inhibited formation of the caudal vein plexus, a bone morphogenic protein-responsive process, an effect rescued by ectopic SMAD1 expression. In mice, miR-26a overexpression inhibited EC SMAD1 expression and exercise-induced angiogenesis. Furthermore, systemic intravenous administration of an miR-26a inhibitor, locked nucleic acid-anti-miR-26a, increased SMAD1 expression and rapidly induced robust angiogenesis within 2 days, an effect associated with reduced myocardial infarct size and improved heart function.

Conclusions: These findings establish miR-26a as a regulator of bone morphogenic protein/SMAD1-mediated EC angiogenic responses, and that manipulating miR-26a expression could provide a new target for rapid angiogenic therapy in ischemic disease states.

Keywords: angiogenesis effect; endothelial cells; microRNAs; myocardial infarction.

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Conflict of interest statement

Disclosures

Mark W. Feinberg, Basak Icli, and The Brigham and Women’s Hospital have a patent pending related to the work that is described in the present study. The other authors report no conflicts.

Figures

Figure 1

Figure 1. MicroRNA (miR)-26a is regulated by proangiogenic stimuli and inhibits cell growth

A, Real-time quantitative polymerase chain reaction (qPCR) analysis of miR-26a expression in response to vascular endothelial cell growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs). *P<0.005 compared with control (Ctrl). B, Real-time qPCR analysis of miR-26a and miR-26b in HUVECs. *P<0.001. C, Circulating miR-26a levels are increased in plasma from human subjects with acute coronary syndrome (n=14) compared with subjects with normal coronary angiograms (n=16). *P<0.05 compared with normal coronary angiogram. D, Circulating miR-26a levels are increased in plasma of mice after 45 minutes of ischemia/reperfusion of the left anterior descending artery (n=4 per group). *P<0.05 compared with sham. HUVECs transfected with miR negative control (NSm), miR-26a mimics (miR-26am), miR inhibitor negative control (NSi), or miR-26a inhibitor (miR-26ai) were subjected to cell growth assays (E; n=6 per group). *P<0.05 compared with NSm or NSi; **P<0.001 compared with NSm or NSi; or cell cycle profiling (F) by propidium iodide staining and fluorescence activated cell sorting (data representative of n=3 experiments). *P<0.001 compared with NSm (G0/G1) or NSi (G0/G1).

Figure 2

Figure 2. MicroRNA (miR)-26a inhibits proangiogenic functions in endothelial cells (ECs) in vitro and in vivo

A and B, Human umbilical vein ECs (HUVECs) transfected with miR negative control (NSm), miR-26a mimics (miR-26am), miR inhibitor negative control (NSi), or miR-26a inhibitor (miR-26ai) were subjected to tube-like network formation in matrigel (A; n=6 per group) or admixed in matrigel plugs (B) placed subcutaneously in nude mice (n=5 per group). CD31 staining was examined in matrigel plugs 1 week later. *P<0.05 compared with NSm; **P<0.05 compared with NSi. Scale bars, 500 μm (upper), 25 μm (lower). EC migration (C) or aortic ring sprouting (D) was examined in response to transfection with NSm, miR-26am, NSi, or miR-26ai. Transwell Boyden chambers were used for EC migration (C) with the indicated growth factors (n=6 per group). *P<0.001 compared with NSm; **P<0.001 compared with NSi. D, Sprouting distance was measured from n=4 to 6 aortic rings per group. *P<0.05 compared with NSm; **P<0.05 compared with NSi. Scale bar, 125 μm.

Figure 3

Figure 3. SMAD1 is a bona fide target of microRNA (miR)-26a in ECs

A, Protein expression of SMAD1 was examined by Western blotting after human umbilical vein endothelial cells (HUVECs) were transfected with miR negative control (NSm), miR-26a mimics (miR-26am), miR inhibitor negative control (NSi), or miR-26a inhibitor (miR-26ai). B, Protein expression of SMAD family members in HUVECs was determined by Western blotting using antibodies to SMAD1, SMAD2, SMAD4, SMAD7, and β-actin (n=3–5 experiments). C and D, Luciferase activity of SMAD1-3′-untranslated region (UTR) normalized to β-galactosidase was quantified in HUVECs transfected with NSm, miR-26am, NSi, or miR-26ai or stimulated with vascular endothelial cell growth factor (VEGF) or tumor necrosis factor (TNF)-α for 6 hours (n=3 experiments). E, Microribonucleoprotein immunoprecipitation analysis of enrichment of SMAD1 mRNA in HUVECs transfected with NSm or miR-26am. *P<0.01. Real-time quantitative polymerase chain reaction was performed to detect SMAD1 (left) or karyopherin alpha 4 (KPNA4) (right). F, HUVECs transfected with NSm, miR-26am, or with miR-26am in the absence or in the presence of lentiviral SMAD1 lacking its 3′-UTR were subjected to cell growth assays (n=4 replicates per condition). *P<0.005 compared with NSm; **P<0.0005 compared with miR-26am. G–I, HUVECs were transfected with siRNA to SMAD1 or scrambled control (Ctrl) siRNA. G, Protein expression was determined by Western analysis using antibodies to SMAD1, Id1, and β-actin (n=2 experiments). H, Tube-like network formation was quantified in matrigel. *P<0.005 (n=6 replicates per condition). I, ECs were subjected to cell growth assays. *P<0.01. Scale bar, 100 μm. Results are representative of n=3 replicates per group and 2 independent experiments. All data represent mean±SEM.

Figure 4

Figure 4. MicroRNA (miR)-26a regulates the expression of downstream bone morphogenic protein (BMP)/SMAD1 signaling in endothelial cells (ECs)

A, Human umbilical vein ECs (HUVECs) transfected with miR negative control (NSm), miR-26a mimics (miR-26am), miR inhibitor negative control (NSi), or miR-26a inhibitor (miR-26ai) (B) were subjected to Western analysis using antibodies to Id1, p21, p27, SMAD1, and β-actin (n=3–5 experiments). C, HUVECs transfected with NSm or miR-26am were treated in the presence or in the absence of BMP9 (0.1 ng/mL) for 2 hours and subjected to Western analysis using antibodies to SMAD1, phosphorylated-SMAD1 (p-SMAD1), and β-actin (n=2 experiments). D, HUVECs were cotransfected with the Id1 promoter along with NSm or miR-26am in the presence or in the absence of BMP9 (0.1 ng/mL) and subjected to luciferase reporter assays (n=3 experiments). *P<0.05 compared with NSm; All data represent mean±SEM.

Figure 5

Figure 5. MicroRNA (miR)-26a regulates caudal vein plexus formation, a bone morphogenic protein (BMP)-responsive process, in zebrafish

Tg(Flk1:EGFP) zebrafish embryos were injected with miR negative control (NSm) or miR-26a mimics (miR-26am) in the presence or in the absence of SMAD1. A, Vasculature of Tg(flk:EGFP) zebrafish embryos was imaged by immunofluorescence confocal microscopy. Inset highlights region of interest for caudal vein plexus. Scale bars, first panel 20 μm, second panel 10 μm, third panel 5 μm. B, The formation of the caudal vein plexus, a BMP-responsive region, was quantified 48 hours after fertilization on a scale of 1 to 10 (n=13 per group). * P<0.001 and (C) vessel density was quantified using ImageJ 1.41. *P <0.005 compared with NSm; ** P<0.001 compared with miR-26am. All data represent mean±SEM.

Figure 6

Figure 6. Inhibition of MicroRNA (miR)-26a increases angiogenesis, decreases infarct size, and improves left ventricular (LV) function in a mouse model of acute myocardial infarction (MI)

A, After a single tail-vein injection in mice of LNA-anti–miR-26a (miR-26ai; 24 mg/kg) or scrambled nonspecific control LNA-anti-miRs (NSi; n=11–12 per group) on day 0, mice underwent acute MI consisting of 45 minutes of ischemia and reperfusion of the left anterior descending artery (LAD) and infusion of fluorescent microbubbles on day 1. B, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining (top) demonstrates areas of infarct in the left ventricle. MI size was normalized to the area at risk. *P <0.05 compared with NSi. Angiogenesis was quantified by CD31 (C) or isolectin staining (D) in sections from the entire LV on day 2. *P<0.05 compared with NSi. Scale bars, 500 μm in (C) and 100 μm in (D). E, LVEF (LVEF) was measured by echocardiography on days 2 and 8. *P<0.05 compared with NSi.

Figure 7

Figure 7. Overexpression of microRNA (miR)-26a inhibits exercise-induced angiogenesis in mice

A, Mice were tail-vein injected with nonspecific scrambled control (NSm) or miR-26am (1 nmol) as indicated for the course of 8 days of defined exercise (wheel-running). B, Representative immunofluorescent staining of CD31 (in red), SMAD1 (in green), Ki67 (in light green), and 4′,6-diamidino-2-phenylindole (DAPI) (in blue) of the quadricep muscles are shown. Scale bars, 50 μm. Quantification of the number of cells staining for CD31 (C), SMAD1 colocalized with CD31 (yellow; D), and Ki67 colocalized with DAPI (E) are shown. * P <0.05 compared with NSm; **P<0.01 compared with NSm. F and G, RNA from the quadriceps muscle was harvested for quantitating the expression of miR-26a (Online Figure VIB), SMAD1 (F), and p21 (G) by real-time quantitative polymerase chain reaction. *P<0.05 compared with NSm. All data represent mean±SEM.

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