**Contents**


## **Yoko Ono and Hidemasa Bono**

Multi-Omic Meta-Analysis of Transcriptomes and the Bibliome Uncovers Novel Hypoxia-Inducible Genes Reprinted from: *Biomedicines* **2021**, *9*, 582, doi:10.3390/biomedicines9050582 ............ **177**

## **About the Editor**

**Kiichi Hirota** is a member of the Institute of Biomedical Science, Head of the Department of Human Stress Response Science, and a professor at Kansai Medical University, Hirakata, Japan. After receiving a Ph.D. in redox biology in the laboratory of Junji Yodoi, Institute for Virus Research, Kyoto, Japan, he started a career as a physician–scientist at Kyoto University Hospital. Then, he was involved in the search for hypoxia biology in the laboratory of Gregg L. Semenza, Johns Hopkins University. As a board-certified anesthesiologist and physician-scientist, he is involved in critical care medicine from the perspective of oxygen metabolism in his own laboratory at Kansai Medical University, Hirakata, Japan.

## **Preface to "Hypoxia-Inducible Factors: Regulation and Therapeutic Potential"**

The role of oxygen as an essential molecule in our survival as mammals is indisputable.

The Special Issue Editor is an anesthesiologist. For anesthesiologists and intensivists, an important goal of patient managemen<sup>t</sup> is to maintain oxygen homeostasis in patients admitted to the operating room or intensive care unit. For this purpose, it is essential to understand the organism's strategies against oxygen imbalance, especially concerning oxygen deprivation (hypoxia). Adaptation to hypoxia and the maintenance of oxygen homeostasis involves a wide range of responses that occur at different tissue levels in the body. On the other hand, oxygen produces cellular damage through the production of reactive oxygen species. Thus, living organisms are built on a precarious balance surrounding oxygen.

Reactive oxygen species and hypoxic conditions produce a signal in the body. In other words, the constantly fluctuating partial pressure of oxygen serves as a cue for the biological response, and a system for rapid response was constructed in the process of evolution. In this way, an in vivo system that senses the dysregulation of oxygen metabolism was developed, and attempts were made to understand this system through comprehensive gene expression analysis. Various endogenous and exogenous stimuli generate reactive oxygen species (ROS), which cause oxidative stress to the organism. Due to the ease of experimental techniques, gene expression studies have accumulated a vast amount of data. However, research on hypoxia using molecular biology has lagged, since molecular oxygen is a gas under atmospheric pressure and normal temperature. The solution to this problem remained an open question until the beginning of the 21st century, when the molecular mechanisms that explain the maintenance and induction of erythropoietin (EPO) expression were discovered. In the early 1990s, the transcription factor hypoxia-inducible factor 1 (HIF-1) was isolated as a factor that explains the molecular mechanism of the maintenance and the induction of erythropoietin (EPO) expression. After cDNA cloning in 1995, a line of research into HIF-1 activation elucidated the molecular mechanism of its regulation of activity in an oxygen partial-pressure-dependent manner. Three enzymes responsible for the hydroxylation of proline residues and one enzyme responsible for the hydroxylation of asparagine residues in the α-subunit of HIF (HIF-α), are now known to play an essential role in this process. There is a consensus that these are cellular "hypoxia sensors". The 2019 Nobel Prize in Physiology or Medicine was awarded to three researchers for their outstanding work in identifying HIF, isolating the molecule, and studying its activation mechanism.

Oxygen is an essential molecule for ATP production in cells, and a scheme is assumed in which the maintenance of biological functions becomes impossible due to the lack of energy caused by its deficiency. It has been thought that a lack of oxygen leads to cell death, the malfunction of biological functions, and death of the individual. However, the classical view of oxygen has been completely revised in the last 20 years. Oxygen is an essential molecule for the maintenance of life. Still, we mammals do not have a mechanism to biosynthesize oxygen in our bodies, and higher organisms such as vertebrates, which are composed of various tissues and organs, are always "deficient" in oxygen. The mainstream view is that the body has evolved mechanisms to respond to the lack of essential molecules or hypoxia and has actively used them to maintain bodily integrity.

This e-book is a collection of research and review papers covering various areas of oxygen biology research that focus on, among other factors, the fundamental understanding of HIF signaling pathways and related gene expression profiling; epigenetic regulation; diagnostics, prognostics, and pharmacogenomic biomarkers; molecular targets driving the regulation of human physiology and pathophysiology; clinical trials with new agents; and validation in animal models.

> **Kiichi Hirota** *Editor*

## *Editorial* **Special Issue: Hypoxia-Inducible Factors: Regulation and Therapeutic Potential**

**Kiichi Hirota**

> Department of Human Stress Response Science, Institute of Biomedical Science, Kansai Medical University, Hirakata 573-1010, Osaka, Japan; khirota-kyt@umin.ac.jp; Tel.: +81-72-804-2526

Oxygen (O2) is an essential molecule [1] in the production of adenosine triphosphate (ATP) in cells, and a lack of energy due to O2 deficiency makes the maintenance of biological functions and human life improbable. Since oxygen functions as the final electron acceptor in the series of ATP synthesis reactions in conjunction with oxidative phosphorylation in mitochondria, its deficiency causes the oxidation of a series of coenzymes such as nicotinamide and flavin adenine dinucleotide and the reduction in oxygen molecules to water molecules (H2O). Persistent deficiency has been believed to cause to the loss of biological functions, even resulting in death. This classical view of oxygen has been completely revised over the last 20 years. Mammals do not have a mechanism for biosynthesizing oxygen in their bodies. In higher organisms such as vertebrates, which possess many organs, oxygen in the body is always "scarce,"; therefore, the dominant view is that organisms have evolved mechanisms to respond to the lack of this essential molecule (hypoxia), and actively use it to maintain body integrity [2,3].

Anatomically complex, higher multicellular organisms are equipped with specialized mechanisms to enable all cells to obtain sufficient oxygen. The respiratory system consists of lungs, which provide oxygen to be transferred to hemoglobin in red blood cells, the diaphragm, other respiratory support muscles, and neuroepithelial cells that sense the partial pressure of oxygen. The cardiovascular system consists of red blood cells, oxygencarrying medium, the heart, the transport engine, blood vessels, and transport channels. The proper development and preservation of these systems require the harmonious expression of thousands of genes. The transcription factor responsible for such gene expression is hypoxia-inducible factor 1 (HIF-1) [3].

In the late 1980s, a team at Johns Hopkins University in Baltimore, USA, searched for an intracellular factor involved in the hypoxia-induced expression of erythropoietin (Epo) and isolated the complementary DNA (cDNA) of a transcription factor in 1995 [4]. This transcription factor was named HIF-1 [5–7]. A closely related gene, hypoxia-inducible factor 2 α (*HIF2A*) or endothelial PAS domain protein 1 (*EPAS1*), was identified and cloned in 1997, followed by hypoxia inducible factor 3 α (*HIF3A*) in 1998 [8]. A series of genes, including those coding for various glycolytic enzymes, glucose transport proteins, vascular endothelial growth factor, and hematopoietic factor Epo, are regulated by HIFs at the transcriptional level. Therefore, HIFs play a role in the activation of "hypoxia-inducible" genes, and we can refer to the term "HIF" as "Highly Involved Factor" [9].

In 2019, the Nobel Prize in Physiology or Medicine was awarded to three researchers for outstanding achievements in this field [10–12].

In this Special Issue, we invited research and review papers in various areas of oxygen biology research that focused on the fundamental understanding of HIF signaling pathways and related gene expression profiling, as well as pharmacogenomic biomarkers, molecular targets driving the regulation of human physiology and pathophysiology, and validation in animal models. As a result, we published six original papers and three review articles in this Special Issue [1,13–20].

Changes in gene expression in response to hypoxic stimuli were studied, with the discovery of hypoxia response systems represented by HIFs. Bono et al. performed a

**Citation:** Hirota, K. Special Issue: Hypoxia-Inducible Factors: Regulation and Therapeutic Potential. *Biomedicines* **2021**, *9*, 1768. https:// doi.org/10.3390/biomedicines9121768

Received: 26 October 2021 Accepted: 29 October 2021 Published: 25 November 2021

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meta-analysis of RNA-sequencing data from hypoxic transcriptomes archived in public databases. In addition, meta-analyzed hypoxic transcriptome data were integrated with public chromatin immunoprecipitation-sequencing data on the known human HIFs, HIF-1, and HIF-2, to provide insights into hypoxia-responsive pathways involving direct transcription factor binding. This study serves as a useful resource for hypoxia research [13]. However, Bono and Ono hypothesized that some hypoxia-responsive genes might not have ye<sup>t</sup> been discovered, hiding behind famous genes. Furthermore, they searched for novel hypoxia-responsive genes in a data-driven manner by utilizing biodigital transformation [19], where data accumulated in a public database (DB) were used for research. They constructed a meta-analysis method to evaluate variations in the expression levels of all genes collected from public databases, before and after hypoxic stimulation, and discovered several novel hypoxia-responsive genes using a multi-omics analysis that integrated information on all genes in the article database. They also created a data-driven meta-analysis method to evaluate alterations in the expression information (transcriptome) of all genes with and without hypoxic stimulation collected from public databases. Thus, they discovered several novel hypoxia-responsive genes through a multi-omics analysis that integrated the collected transcriptome information with genes published in all papers (bibliome) in the public DB.

The secretome is expected to be useful in biomarker discovery because it contains abundant proteins and fragments of membrane proteins secreted and released by cancer cells, unlike blood samples, which do not contain secretions from various tissues.

The term "secretome" is a general term that includes not only soluble proteins released from cells via the endoplasmic reticulum (ER)-Golgi pathway, but also extracellular matrix proteins and cleaved fragments of membrane proteins. Moog et al. have shown that the properties of the secretome are affected by hypoxia in a series of studies [16–18].

It has been reported that HIF-1 regulates the induction of matrix metalloproteinases (MMPs). Wei et al. found that xanthine oxidase-derived reactive oxygen species (ROS) induced MMP-3, MMP-10, and MMP-13 in mouse macrophages upon hypoxic exposure. Induction was not inhibited by HIF-1 α-deficient macrophages, but was HIF-1-independent. Additionally, induction was found to be inhibited by febuxostat, a xanthine oxidase inhibitor. Febuxostat administration is a potential therapeutic option for disease managemen<sup>t</sup> in atherosclerotic patients [20].

The role of oxygen metabolism in the regulation of inflammation and immune responses has attracted attention. Humoral factors, including cytokines and chemokines, and physical stimuli such as heat and tissue breakdown induce ROS and generate oxidative stress, while impaired blood vessels, impaired blood flow, increased oxygen consumption by infiltrating cells, and impaired oxygen diffusion due to edema reducing the oxygen concentration in tissues. Inflammatory cells, which work "in the field" of inflammation, adapt to these changes in the oxygen environment to maintain cellular functions and contribute to biological defense and homeostasis [21,22]. Chen and Gaber summarized the effects of physiological and pathophysiological hypoxia on innate and adaptive immune activity. We provide an overview on the control of immune response by cellular hypoxiainduced pathways, with a focus on the role of HIFs, and discuss the opportunity to target hypoxia-sensitive pathways for the treatment of cancer and autoimmunity [14].

HIF plays an essential role in this process; it is a transcription factor that mediates Epo induction at the transcriptional level under hypoxic conditions. In 2001, cDNA cloning of dioxygenases acting on prolines and asparagine residues, which play essential roles in this process, was reported. HIF-prolyl hydroxylases (PHs) constitute the core molecular mechanism for detecting a decrease in the partial pressure of oxygen, or hypoxia, in the cells, and are known as oxygen sensors [23–26]. In this review, I discuss the process of the molecular cloning of HIF and HIF-PHs, which explains hypoxia-induced Epo expression, the development of HIF-PH inhibitors that artificially or exogenously activate HIF by inhibiting HIF-PH, and the significance and implications of medical intervention using HIF-PH inhibitors [15].

We hope that this Special Issue will reflect the current exciting researches concerning HIFs and their applications in medicine and health science.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
