Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis
Goutam Ghosh, … , Yan Zeng, Sundaram Ramakrishnan
Goutam Ghosh, … , Yan Zeng, Sundaram Ramakrishnan
Published October 25, 2010
Citation Information: J Clin Invest. 2010;120(11):4141-4154. https://doi.org/10.1172/JCI42980.
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Research Article

Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis

Abstract

Adaptive changes to oxygen availability are critical for cell survival and tissue homeostasis. Prolonged oxygen deprivation due to reduced blood flow to cardiac or peripheral tissues can lead to myocardial infarction and peripheral vascular disease, respectively. Mammalian cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for what we believe is a novel mechanism regulating HIF-1α levels in isolated human ECs during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in ECs. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 was regulated by PU.1-dependent transactivation. PU.1 levels were increased in hypoxic endothelium by RUNX-1 and C/EBPα. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by a specific morpholino. The rodent homolog of human miR-424, mu-miR-322, was significantly upregulated in parallel with HIF-1α in experimental models of ischemia. These results suggest that miR-322/424 plays an important physiological role in post-ischemic vascular remodeling and angiogenesis.

Authors

Goutam Ghosh, Indira V. Subramanian, Neeta Adhikari, Xiaoxiao Zhang, Hemant P. Joshi, David Basi, Y.S. Chandrashekhar, Jennifer L. Hall, Sabita Roy, Yan Zeng, Sundaram Ramakrishnan

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Figure 4

Effect of miR-424 on in vitro angiogenesis.

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Effect of miR-424 on in vitro angiogenesis. (A and C) The effects of miR...
(A and C) The effects of miR-424 overexpression under normoxia and silencing by miR-424–morpholino under hypoxia on EC migration were determined by scratch wound assay. Wound closure was determined after 24 hours. Yellow and green lines indicate edges of scratch wounds. Representative photo­micrographs are shown. Migration of miR-424–overexpressing cells under normoxia (A). Migration of miR-424–morpholino–treated cells under hypoxia (C). (B and D) Quantification of cell migration. (B) Normoxia (miR-control or miR-424 overexpression). (D) Hypoxia (control morpholino or miR-424–morpholino). n = 3. (E) Effect of miR-424 on cell proliferation. BrdU incorporation was determined by ELISA. Values represent mean ± SD. n = 3. (F) Representative confocal images of HUVECs stained for the proliferation marker PCNA. HUVEC cultures were transfected with a control morpholino or miR-424–morpholino and grown in media containing 5% FBS and VEGF (50 ng/ml) under hypoxia. Cells were fixed and stained with anti-PCNA antibody and developed by Alexa Fluor 488–labeled secondary antibody conjugate. Also stained for F-Actin (rhodamine phalloidin: red) and nuclei (DAPI: blue). Scale bar: 20 μm. (G) Top: Representative photomicrographs of capillary tube formation of HUVECs transfected with miR-control or miR-424. Tube formation was scored after 18 hours. Bottom: Tube formation capacity of HUVECs transfected with control morpholino or miR-424–morpholino. The morpholino effect was investigated under hypoxia. Total magnification, ×100. (H) Morphometric analyses of capillary tube length from 3 independent experiments. Values represent mean ± SD. **P ≤ 0.02.