Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis
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
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
View: Text | PDF
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

×

Figure 5

Effect of miR-424 on in vivo angiogenesis.

Options: View larger image (or click on image) Download as PowerPoint
Effect of miR-424 on in vivo angiogenesis. (A) HUVECs were transduced wi...
(A) HUVECs were transduced with either GFP-positive miR-control– or GFP-positive miR-424–expressing retrovirus. Transduced GFP-positive HUVECs were admixed with ovarian cancer cells in Matrigel and subcutaneously implanted into athymic mice, harvested after 7 days, and processed for vessel staining. Mouse ECs (red), HUVECs (green), and chimeric vessels are shown in merged images. Scale bars: 50 μm. (B) Metamorph analysis of colocalized pixels of mouse endothelium (red) and HUVECs (green) is shown. Five animals per group and 2 sections of Matrigels (100 μm from the surface) from each sample were analyzed. Ten frames from each section were used to determine the colocalized pixels. (C) Mu-miR-322 levels in rat cardiac tissues from sham-operated and LAD artery–ligated animals. Peri-infarct (P) and remote (R) areas are shown in the schematic diagram. (D) Photomicrographs show representative images of blood vessels and HIF-1α from the gastrocnemius muscle sections from sham-operated and femoral artery–ligated animals. Vessels were stained with tomato-lectin (red) and HIF-1α (green). Arrowheads indicate colocalization of HIF-1α in blood vessels. Scale bars: 40 μm. (E) Formalin-fixed tissue sections from sham-operated and ligated animals were used for in situ hybridization to determine changes in mu-miR-322. Sections were hybridized with miR-322–specific LNA probe (red). The same sections were also stained for CUL2 (green) and nucleus (blue). Scale bars: 10 μm. (F) q-PCR data of mu-miR-322 levels in the gastrocnemius muscle from sham-operated and ligated animals (n = 3). Values represent mean ± SD. *P < 0.05.