MicroRNA-34a-Dependent Attenuation of Angiogenesis in Right Ventricular Failure

Abstract

Background: The right ventricle (RV) is at risk in patients with complex congenital heart disease involving right-sided obstructive lesions. We have shown that capillary rarefaction occurs early in the pressure-loaded RV. Here we test the hypothesis that microRNA (miR)-34a, which is induced in RV hypertrophy and RV failure (RVF), blocks the hypoxia-inducible factor-1α-vascular endothelial growth factor (VEGF) axis, leading to the attenuated angiogenic response and increased susceptibility to RV failure.

Methods and results: Mice underwent pulmonary artery banding to induce RV hypertrophy and RVF. Capillary rarefaction occurred immediately. Although hypoxia-inducible factor-1α expression increased (0.12±0.01 versus 0.22±0.03, P=0.05), VEGF expression decreased (0.61±0.03 versus 0.22±0.05, P=0.01). miR-34a expression was most upregulated in fibroblasts (4-fold), but also in cardiomyocytes and endothelial cells (2-fold). Overexpression of miR-34a in endothelial cells increased cell senescence (10±3% versus 22±2%, P<0.05) by suppressing sirtulin 1 expression, and decreased tube formation by 50% via suppression of hypoxia-inducible factor-1α, VEGF A, VEGF B, and VEGF receptor 2. miR-34a was induced by stretch, transforming growth factor-β1, adrenergic stimulation, and hypoxia in cardiac fibroblasts and cardiomyocytes. In mice with RVF, locked nucleic acid-antimiR-34a improved RV shortening fraction and survival half-time and restored capillarity and VEGF expression. In children with congenital heart disease-related RVF, RV capillarity was decreased and miR-34a increased 5-fold.

Conclusions: In summary, miR-34a from fibroblasts, cardiomyocytes, and endothelial cells mediates capillary rarefaction by suppressing the hypoxia-inducible factor-1α-VEGF axis in RV hypertrophy/RVF, raising the potential for anti-miR-34a therapeutics in patients with at-risk RVs.

Keywords: angiogenesis; congenital heart disease; fibroblasts; heart failure; microRNA; right ventricle.

Figure 1. RV pressure overload is characterized by failure to increase capillarity, and RV failure is characterized by capillary rarefaction.

A, Cardiac sections from mice with increasing severity of RV hypertrophy and RV failure were stained with WGA (green), a cell membrane marker and CD31 (red), an endothelial cell marker and compared with sham‐operated mice. B, Cell area increased progressively with increasing severity of PS. C, Capillary‐to‐myocyte ratio does not increase in mild or moderate PS and is decreased in RV failure. D, Capillary‐to‐cardiomyocyte cell area decreased. N=3 to 8/group. Scale bars=100 μm. PS indicates pulmonary stenosis; RV, right ventricle; RVF, RV failure; and WGA, wheat germ agglutinin. Data are presented as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 using ANOVA.

Figure 2. RV failure is characterized by upregulation of Hif‐1α, downregulation of Vegf, and upregulation of miR‐34a.

A, Total Hif‐1α protein expression increases with increasing severity of RV hypertrophy and RV failure. B, Hif‐1α protein expression is increased in both cytosol and to a larger degree in the nucleus. C and D, Vegf A and Vegf B protein expression decrease most dramatically with RV failure despite persistent Hif‐1α upregulation; N=6/group. E, Hif‐1α target genes HO‐1 and Gapdh are upregulated, Epo is unchanged, and Vegf is downregulated in RV failure, F, Hif‐1α target miRs 199a, 214, and 210 are upregulated in RV failure. N=4/group. G, miR‐34a expression increases with increasing severity of RV hypertrophy and RV failure; H, miR‐34a is highly expressed in fibroblasts and to a lesser extent in cardiomyocytes and endothelial cells in RV failure. N=4/group. Epo indicates erythropoietin; Gapdh, glyceraldehyde phosphate dehydrogenase; Hif, hypoxia inducible factor; HO‐1, hemo oxygenase; LV, left ventricle; miR, microRNA; PS, pulmonary stenosis; RV, right ventricle; RVF, RV failure; and Vegf, vascular endothelial growth factor. Data are presented as mean±SEM. *P<0.05, **P<0.01, ***P<0.001.

Figure 3. miR‐34a impairs endothelial cell function by decreasing Hif‐1α and Vegf signaling.

Lipofectamine‐based miR‐34a was transfected in human microvascular cardiac endothelial cells and led to (A and B) increased cell senescence (β‐galactosidase activity in green, arrow), (scale bars=100 μm) via (C and D) downregulation of Sirt 1 protein expression; (E and F) decreased tube formation, (scale bars=1000 μm) via (G through L) downregulation of Hif‐1α, Vegf A, Vegf B, and Vegf receptor 2 phosphorylation protein expression. N=3–4/group. Hif indicates hypoxia inducible factor; miR, microRNA; Sirt, sirtulin; and Vegf, vascular endothelial growth factor. Data are presented as mean±SEM. *P<0.05, **P<0.01 using t test.

Figure 4. miR‐34a is upregulated by stretch, TGF‐β1, isoproterenol, and hypoxia.

A, Stretch upregulates miR‐34a in cardiac fibroblasts but not in cardiomyocytes and cardiac endothelial cells. B, Isoproterenol stimulation (100 μmol/L×24 hours) upregulates miR‐34a in cardiac fibroblasts and cardiomyocytes but not in cardiac endothelial cells. C, Combined TGF‐β1 (5 ng/mL×24 hours) and isoproterenol (100 μmol/L×24 hours) stimulation of cardiac fibroblasts upregulates miR‐34a more than either agent alone (N=3–4/group). D, 1% Hypoxia exposure for 24 hours increases miR‐34a expression in cardiomyocytes only. E, Schematic of proposed mechanism of miR‐34a upregulation in right ventricular pressure overload. Hif indicates hypoxia inducible factor; miR, microRNA; Sirt, sirtulin; TGF, transforming growth factor; and Vegf, vascular endothelial growth factor. Data are presented as mean±SEM. *P<0.05, ***P<0.001.

Figure 5. Anti‐miR‐34a improves survival and rescues capillarity in RV failure.

Anti‐miR‐34a (A) suppresses miR‐34a levels, (B) improves RVOT SF, and (C) improves survival half time by 44% (P<0.02), N=8–9/group. Anti‐miR‐34a treatment (D and E) increases capillarity (CD31 staining of capillaries as brown dots, arrows) and (F, G) increases Vegf expression, N=5/group. (H) Plasma miR‐34a expression is increased at all stages of mild and moderate PS and RVF while plasma miR‐34a expression returns to baseline with anti‐miR treatment. miR indicates microRNA; PS, pulmonary stenosis; RV, right ventricle; RVF, right ventricular failure; RVOT SF, right ventricular outflow tract shortening fraction; and Vegf, vascular endothelial growth factor. Scale bars=50 μm. Data are presented as mean±SEM. *P<0.05, **P<0.01 using ANOVA and Kaplan–Meier for survival analysis.

Figure 6. Children with RV failure show dysregulation in angiogenesis.

A and B, Capillarity decreases with RV failure (CD31 staining of capillaries as brown dots, arrow). C, RV miR‐34a expression is increased with RV failure in children, N=3/group. D and E, Representative blots and protein expression quantification showing that Hif‐1α protein expression is unchanged in RV failure but Vegf A, Vegf B, and Sirt 1 protein expression is decreased with RV failure. N=3–4/group. Hif indicates hypoxia inducible factor, miR, microRNA; PS, pulmonary stenosis; Sirt, sirtulin; RV, right ventricle; RVF, RV failure; and Vegf, vascular endothelial growth factor. Scale bars=100 μm. Data are presented as mean±SEM. *P<0.05, **P<0.01.

Figure 7. Summary of the dysregulation of the microvasculature in RV failure due to congenital heart disease in children.

HIF indicates hypoxia inducible factor; miR, microRNA; RV, right ventricle; SIRT, sirtulin; and VEGF, vascular endothelial growth factor.