The decline of oxygen tension in the tissues below the physiological demand leads to the hypoxic adaptive response. This physiological consequence enables cells to recover from this cellular insult. Understanding the cellular pathways that mediate recovery from hypoxia is therefore critical for developing novel therapeutic approaches for cardiovascular diseases and cancer. The master regulators of oxygen homeostasis that control angiogenesis during hypoxia are hypoxia-inducible factors (HIFs). HIF-1 and HIF-2 function as transcriptional regulators and have both unique and overlapping target genes, whereas the role of HIF-3 is less clear. HIF-1 governs the acute adaptation to hypoxia, whereas HIF-2 and HIF-3 expressions begin during chronic hypoxia in human endothelium. When HIF-1 levels decline, HIF-2 and HIF-3 increase. This switch from HIF-1 to HIF-2 and HIF-3 signaling is required in order to adapt the endothelium to prolonged hypoxia. During prolonged hypoxia, the HIF-1 levels and activity are reduced, despite the lack of oxygen-dependent protein degradation. Although numerous protein factors have been proposed to modulate the HIF pathways, their application for HIF-targeted therapy is rather limited. Recently, the miRNAs that endogenously regulate gene expression via the RNA interference (RNAi) pathway have been shown to play critical roles in the hypoxia response pathways. Furthermore, these classes of RNAs provide therapeutic possibilities to selectively target HIFs and thus modulate the HIF switch. Here, we review the significance of the microRNAs on the relationship between the HIFs under both physiological and pathophysiological conditions.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. The initial stage of CVDs is characterized by endothelial dysfunction, defined as the limited bioavailability of nitric oxide (NO). Thus, any factors that interfere with the synthesis or metabolism of NO in endothelial cells are involved in CVD pathogenesis. It is well established that hypoxia is both the triggering factor as well as the accompanying factor in cardiovascular disease, and diminished tissue oxygen levels have been reported to influence endothelial NO bioavailability. In endothelial cells, NO is produced by endothelial nitric oxide synthase (eNOS) from L-Arg, with tetrahydrobiopterin (BH₄) as an essential cofactor. Here, we discuss the mechanisms by which hypoxia affects NO bioavailability, including regulation of eNOS expression and activity. What is particularly important is the fact that hypoxia contributes to the depletion of cofactor BH₄ and deficiency of substrate L-Arg, and thus elicits eNOS uncoupling—a state in which the enzyme produces superoxide instead of NO. eNOS uncoupling and the resulting oxidative stress is the major driver of endothelial dysfunction and atherogenesis. Moreover, hypoxia induces impairment in mitochondrial respiration and endothelial cell activation; thus, oxidative stress and inflammation, along with the hypoxic response, contribute to the development of endothelial dysfunction.

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.

Hypoxia-inducible factors (HIF) are heterodimeric transcription factors that allow cells to adapt and survive during hypoxia. Regulation of HIF1A and HIF2A mRNA is well characterized, whereas HIF3A mRNA regulation and function are less clear. Using RNA-Seq analysis of primary human umbilical vein endothelial cells, we found two isoforms of HIF3A were expressed, HIF3A2 and HIF3A3. Comparing HIF3A expression profiles to HIF1A mRNA during 48 hours of hypoxia revealed that HIF1A message peaked at 4 hours, whereas HIF3A expression increased while HIF1A was decreasing. Given that HIF1A mRNA is regulated by miR-429, we tested miR-429 effects on both HIF3A isoforms and found that they too were regulated by miR-429. Analysis of a HIF-3 target, DNA-damage-inducible transcript 4, a key survival gene, indicated that DDIT4 mRNA is induced by HIF-3 and negatively regulated by miR-429 through miR-429’s actions on HIF3A message. This provides a compelling model for how hypoxia-induced miR-429 regulates the switch between HIF-1 adaptive responses to HIF-3 survival responses by rapidly decreasing HIF1A levels while simultaneously slowing the progression of HIF3A expression until the miR-429 levels drop below normoxic levels. Since HIF-1 drives HIF3A and miR-429 expression, this establishes a regulatory network in which miR-429 plays a pivotal role.

Understanding the cellular pathways that regulate endothelial nitric oxide (eNOS, NOS3) expression and consequently nitric oxide (NO) bioavailability during hypoxia is a necessary aspect in the development of novel treatments for cardiovascular disorders. eNOS expression and eNOS-dependent NO cellular signaling during hypoxia promote an equilibrium of transcriptional and posttranscriptional molecular mechanisms that belong to both proapoptotic and survival pathways. Furthermore, NO bioavailability results not only from eNOS levels, but also relies on the presence of eNOS substrate and cofactors, the phosphorylation status of eNOS, and the presence of reactive oxygen species (ROS) that can inactivate eNOS. Since both NOS3 levels and these signaling pathways can also be a subject of posttranscriptional modulation by microRNAs (miRNAs), this class of short noncoding RNAs contribute another level of regulation for NO bioavailability. As miRNA antagomirs or specific target protectors could be used in therapeutic approaches to regulate NO levels, either by changing NOS3 mRNA stability or through factors governing eNOS activity, it is critical to understand their role in governing eNOS activity during hypoxa. In contrast to a large number of miRNAs reported to the change eNOS expression during hypoxia, only a few miRNAs modulate eNOS activity. Furthermore, impaired miRNA biogenesis leads to NOS3 mRNA stabilization under hypoxia. Here we discuss the recent studies that define miRNAs’ role in maintaining endothelial NO bioavailability emphasizing those miRNAs that directly modulate NOS3 expression or eNOS activity.