eNOS expression and NO release during hypoxia is inhibited by miR-200b in human endothelial cells

Abstract

The nitric oxide (NO) secreted by vascular endothelium is required for the maintenance of cardiovascular homeostasis. Diminished release of NO generated by endothelial NO synthase contributes to endothelial dysfunction. Hypoxia and ischemia reduce endothelial eNOS expression via posttranscriptional mechanisms that result in NOS3 transcript destabilization. Here, we examine whether microRNAs contribute to this mechanism. We followed the kinetics of hypoxia-induced changes in NOS3 mRNA and eNOS protein levels in primary human umbilical vein endothelial cells (HUVECs). Utilizing in silico predictive protocols to identify potential miRNAs that regulate eNOS expression, we identified miR-200b as a candidate. We established the functional miR-200b target sequence within the NOS3 3′UTR, and demonstrated that manipulation of the miRNA levels during hypoxia using miR-200b mimics and antagomirs regulates eNOS levels, and established that miR-200b physiologically limits eNOS expression during hypoxia. Furthermore, we demonstrated that the specific ablation of the hypoxic induction of miR-200b in HUVECs restored eNOS-driven hypoxic NO release to the normoxic levels. To determine whether miR-200b might be the only miRNA that had this effect, we utilized Next Generation Sequencing (NGS) to follow hypoxia-induced changes in the miRNA levels in HUVECS and found 83 novel hypoxamiRs, with two candidate miRNAs besides miR-200b that could potentially influence eNOS levels. Taken together, the data establish miR-200b-eNOS regulation as a first hypoxamiR-based mechanism that limits NO bioavailability during hypoxia in endothelial cells, and show that hypoxamiRs could become useful therapeutic targets for cardiovascular diseases and other hypoxic-related diseases including various types of cancer.Electronic supplementary materialThe online version of this article (10.1007/s10456-018-9620-y) contains supplementary material, which is available to authorized users.

Primary endothelial cell–specific regulation of hypoxia‐inducible factor (HIF)‐1 and HIF‐2 and their target gene expression profiles during hypoxia

“…We used TaqMan One-Step Real-Time PCR Master Mix Reagents (Thermo Fisher Scientific) as previously described (25,26) using the manufacturer's protocol (retrotranscription: 15 min, 48°C). The relative expressions were calculated using the 2 2DDCt method (27) with the TATA-binding protein (TBP) and 18S rRNA genes as reference genes for the mRNA.…”

Targeting mitochondria‐associated membranes as a potential therapy against endothelial injury induced by hypoxia

“…In a broad sense, hypoxia stems from conditions that include exposure to high altitude, the milieu of wounds and burn injuries, areas of rapid tumor growth, and restricted bloodstreams due to ischemic vascular diseases. During pathogenesis, hypoxia acts as a powerful force to perturb endothelial homeostasis, leading to a burst in reactive oxygen species (ROS) production, decrease in nitric oxide (NO) synthesis, promotion of vasoconstrictive substances, and elevated expression of pro‐inflammatory cytokines and adhesion molecules such as intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule 1 (VCAM‐1), which have been thoroughly investigated in the context of hypoxic pulmonary hypertension for decades . Numerous molecular mechanisms are implicated in hypoxia‐induced endothelial injury.…”

“…During pathogenesis, hypoxia acts as a powerful force to perturb endothelial homeostasis, leading to a burst in reactive oxygen species (ROS) production, decrease in nitric oxide (NO) synthesis, promotion of vasoconstrictive substances, and elevated expression of pro-inflammatory cytokines and adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), which have been thoroughly investigated in the context of hypoxic pulmonary hypertension for decades. [3][4][5][6][7][8][9] Numerous molecular mechanisms are implicated in hypoxia-induced endothelial injury. Among these, mitochondria serve not only as energy-producing machines but also as signalling hubs that orchestrate and activate a set of signals such as those associated with redox, metabolism, autophagy, proliferation, inflammation, and intrinsic death pathways.…”

Expression and Significance of MicroRNA155 in Serum of Patients with Cerebral Small Vessel Disease

“…Hypoxia and ischemia reduce endothelial nitric oxide synthetase (eNOS) expression in endothelial cells through post-transcriptional mechanism, resulting in NOS3 transcriptional instability. In this study, we found that miRNA contributes to this mechanism 8) . By studying changes in the expression profile of miRs in ischemic stroke, it was found that the expression level of miR-155 in rat brain tissue was increased 36) , indicating that miR-155 was closely related to ischemic stroke.…”