All animal cells need oxygen for energy production and cannot stay alive without it. Due to this, evolution has provided cells with a molecular-level mechanism that recognises a low oxygen level, or hypoxia, and starts a genetic programme to respond to it. The response to hypoxia involves hundreds of different genes that help cells survive in low-oxygen conditions.
The physiological effects of the response to hypoxia – scientifically referred to as the HIF (hypoxia-inducible factor) response – were already observed in the early 20th century: in a low-oxygen environment, the number of red blood cells and level of haemoglobin increase, for instance. These effects were made use of at places like mountain sanatoriums and high-altitude camps for athletes, even though their cause was not known.
With the development of gene technology, researchers started getting a hint of the mechanisms at the beginning of the 1990s, when they noticed that low oxygen levels led to the activation of certain genes. The next discovery, in 1995, was the transcription factor that induces a response to hypoxia – a protein that activates the response genes as its disintegration stops when the oxygen runs low. In 2001, the “oxygen sensor” of cells was discovered: a family of enzymes whose activity wanes in hypoxia, leading to the stability of the transcription factor.
The latest milestone is a medicine that prevents the activation of the oxygen sensor, i.e. the HIF enzymes. In other words, the medicine makes the cell believe that it suffers from oxygen deprivation, leading to the response to hypoxia. The primary indication for the medicine is severe anaemia: the condition reduces the number of oxygen-carrying red cells, whereas one of the genes activated in the response to hypoxia produces erythropoietin (also known as the doping agent EPO), which stimulates red blood cell production The first marketing authorisations for the medicine were granted in December 2018, and it is already available in China and Japan.
The long journey that started from mountain sanatoriums will culminate in this year’s Nobel Prize. The University of Oulu also joined in along the way.
Oulu was the first place in the world to manufacture oxygen sensor enzymes
The University of Oulu’s role in hypoxia research dates back four decades to a different subject, Academy Professor Kari Kivirikko’s collagen research. The research projects led by Kari Kivirikko on collagen, the most common protein in connective tissues, raised Oulu to the top of the field as early as in the 1970s. In the 90s, Peppi Karppinen and Johanna Myllyharju also worked on the subject.
“Oulu had top expertise in the enzyme that regulates the formation of collagen,” says Johanna Myllyharju, Professor of Molecular Biology. “When the first oxygen sensor enzymes were detected in Oxford in 2000, they were found to be surprisingly similar to collagen enzymes. The people in Oxford knew that collagen enzymes had been researched in Oulu, and they asked us to test whether they happened to influence the response to hypoxia.”
The result was negative – and led to a breakthrough. “It was the starting point for their search for other similar enzymes. And in 2001, they found a whole new family of enzymes.”
In Oulu, knowledge of corresponding collagen enzymes was immediately harnessed for research into the response to hypoxia. Soon, Johanna Myllyharju and Peppi Karppinen became the first in the world to manufacture HIF enzymes as recombinants – by cloning the human gene that produces the enzyme and adding it to an insect cell. This enabled the production of the enzymes and research into their properties. In addition, several researchers and publications showed that the activity of HIF enzymes dwindles as the oxygen runs low, resulting in the response to hypoxia.
Both achievements offered tools for pharmaceutical development. The medicine now introduced into the market that produces the response to hypoxia, the HIF prolyl-hydroxylase inhibitor, has been developed by American company FibroGen. FibroGen is also linked with Oulu and Kari Kivirikko.
“The company founder wanted to develop medicines for connective tissue diseases and contacted Kivirikko in 1993, since he was the best expert in the collagen enzymes, which guide the formation of connective tissues. This resulted in the University of Oulu signing a research collaboration agreement with the new firm, and FibroGen became a partial funder of our research projects in 1994,” Johanna Myllyharju says.
Collagen enzymes were at the core of the collaboration with FibroGen until 2001. After that, they were joined by HIF enzymes. FibroGen already had collagen prolyl-hydroxylase inhibitors, which the researchers in Oulu started testing on the HIF enzymes, giving FibroGen a head start on other pharmaceutical manufacturers.
With these merits, the University of Oulu has a strong position in the international hypoxia research community.
“Even others think we are at the forefront,” says Peppi Karppinen, now the Dean of the Faculty of Biochemistry and Molecular Medicine. “We have methods that are not available to others, such as measuring the activity of the enzymes and producing recombinant enzymes.”
There are approximately 50 hypoxia researchers working in the faculty in the research teams of Johanna Myllyharju, Peppi Karppinen and Thomas Kietzmann. Karppinen and Myllyharju have also been cooperating with the Nobel Prize recipients: Peter Ratcliffe (Oxford), William Kaelin (Harvard) and Gregg Semenza.
“I phoned Kaelin to congratulate him, and he also congratulated us,” says Peppi Karppinen, who is going to Stockholm for the Nobel after-party. She thinks that pharmaceutical development is the reason why the Nobel Prize came this year. Kaelin’s and Ratcliffe’s teams published their findings simultaneously in 2001, whereas the findings in the 1990s were mostly made by Semenza.
Hopes for derivatives of anaemia medication to help with other diseases
The next research objectives have to do with more extensive understanding of the response to hypoxia and new medication.
“The hypothesis is that there are more enzymes affecting the response – but it may also be a question of something else,” Professor Myllyharju says.
In addition to anaemia, hypoxia is also linked with many other diseases. “In infarcts, for example, there is oxygen deprivation in the affected tissues. Blood vessels are damaged in traumas, such as fractures, and oxygen deprivation also occurs in inflammatory states. Firm tumours are usually hypoxic, since their cell division exceeds the capacity of forming new blood vessels.”
The latter is a special case, since the response to hypoxia mostly benefits the organism, but the ability of cancer cells to survive in low-oxygen conditions may also be fatal. However, there is no proof of the response to hypoxia causing cancer or promoting its growth.
“The risk was taken into account in the development of anaemia medication from the outset,” Johanna Myllyharju says. “Medication is also being developed to deaden the response to hypoxia in cancer cells. This is targeted at different parts of the response than our enzyme inhibitors are.”
Also, there are hopes for derivatives of anaemia medication to help with ischaemia (local restriction in blood supply to tissues, caused by infarct or artery constriction, for instance), inflammatory diseases as well as the treatment of obesity and the metabolic syndrome.
“The response to hypoxia has inflammation-restraining effects, which was already indirectly known about at mountain sanatoriums,” Peppi Karppinen says. “It also changes metabolism: the amount of harmful cholesterol decreases, and when a hypoxic cell produces energy without oxygen, only a fraction of the usual energy is produced, which is likely to lead to a weight-loss effect.”
At the moment, these pharmaceutical applications are only being tested on cells or animals. Nevertheless, the development has been quick – and surprising, as Johanna Myllyharju points out.
“When Kari Kivirikko started researching collagen enzymes in the 1960s, nobody expected that a whole new family of enzymes would be discovered 40 years later, with a similar mechanism but affecting a different biological phenomenon. This is good proof of the significance of basic research.”
Text: Jarno Mällinen
Photo: Professor Johanna Myllyharju (on the left) and Professor Peppi Karppinen. (Kulmakuvaamo/Seija Leskelä )
Last updated: 10.12.2019