key: cord-0862942-0f8lw4ul authors: Hertzog, Radu Gabriel; Bicheru, Nicoleta Simona; Popescu, Diana Mihaela; Călborean, Octavian; Catrina, Ana-Maria title: Hypoxic preconditioning – a non-pharmacological approach in COVID-19 prevention date: 2020-11-26 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2020.11.181 sha: bcc31323a9810c3f0286d6a0d04dcbd0aa69a1c0 doc_id: 862942 cord_uid: 0f8lw4ul Hypoxia is defined by low oxygen concentration in organs, tissues and cells. Maintaining oxygen homeostasis represents the essential cellular metabolic process for the structural integrity of tissues in different pathological conditions, including SARS-CoV-2 infection. Considering the role of hypoxia–inducible factor-1 (HIF-1) as regulator of cellular response to hypoxia and its involvement in angiogenesis, erythropoiesis, glucose metabolism, inflammation, we propose hypoxic preconditiong (HPC) as a novel prevention therapeutic approach on healthy contacts of COVID-19 patients. To date, several studies revealed the benefic effects of hypoxic preconditioning in ischemia, kidney failure and in pulmonary function recovery of lung surgery patients. HPC increases the expression of factors that promote cell survival and angiogenesis, induces an anti-inflammatory outcome, triggers coordinated hypoxia responses that promote erythropoiesis, and mobilizes the circulating progenitor cells. Futhermore, the mesenchymal stem cells (MSC) exposed to HPC show improvement of their regenerative capacities, and increases the effectiveness of stem cell therapy in different pathologies, including COVID-19. In conclusion, HPC should be considered an approach with beneficial outcomes and without significant side effects when the organism is severely exposed to the same stressor. HPC appears as a trigger to mechanisms that improve and maintain tissue oxygenation and repair, a main goal in different pathologies, including COVID-19 or other respiratory conditions. The oxygen concentration between 20-21% is definitory for normoxia. On the contrary, hypoxic conditions are reached at low oxygen levels in organs, tissues and cells. The cellular response to hypoxia is mediated by the hypoxia-inducible factor-1 (HIF-1) transcription factor, which represents the key regulator of oxygen homeostasis, promoting cellular adaptation to reduced oxygen availability (Semenza, 2006; Wenger et al., 2005) . It has been shown that in response to decreased oxygen supply, HIF-1 regulates the transcription of a multitude of genes whose protein products promote different aspects of hypoxic adaptation, including angiogenesis, erythropoiesis, oxygen transport (increased oxygen delivery), glucose metabolism and metabolic adaptation, vascular tone, cell proliferation and survival. VEGF, erythropoietin, glucose transporters, glycolytic enzymes, NO and adenosine are some of the key targets of HIF-1α (Semenza, 2006; Wenger et al., 2005) . Hypoxia can be caused by a variety of conditions, from intense physical effort and high altitude to lung disease, inflammation, infarction and carcinogenesis (Kumar and Choi, 2015) . Maintaining oxygen homeostasis is the key in cellular metabolic processes necessary to allow the structural integrity of tissues. Tissue hypoxia appears due to hypoxemia, oxygen delivery deficiencies or deficiencies in the oxygen consumption at the cellular level (MacIntyre, 2014) . The improvement and maintainence of tissue oxygenation is an important goal for the future in different pathologic conditions, including COVID-19. In the present paper, the very likely beneficial preventitive effects of hypoxic preconditioning on healthy contacts of COVID-19 patients are discussed. The virus entry into host cells is mediated by host serine transmembrane type 2 protease (TMPRSS2), which facilitates viral entry by cleaving ACE2 and activating the S protein of SARS-CoV-2. ACE2 and TMPRSS2 are primarily expressed in type II alveolar epithelial cells (Wiersinga et al., 2020) . The body's inflammatory response to COVID-19 infection consists of an intense release of pro-inflammatory cytokines, a phenomenon described as a "cytokine storm". Scientific data analyzing cytokine profile in patients diagnosed with COVID-19 have shown that the "cytokine storm" is directly related to lung damage and insufficiency in organs (Huang et al., 2020; Ragab et al., 2020; Ruan et al., 2020) . The occurrence of the "cytokine storm" is caused by a rapid increase in pro-inflammatory cytokines (IL-6, IL-1, TNF-α and interferon), leading to acute lung lesions or in more severe forms, the onset of acute respiratory failure syndrome (ARDS) (Shimizu, 2019) . ARDS is associated with low levels of oxygen saturation, which is a major cause of mortality with COVID-19 (N. Chen et al., 2020) . On the other hand, comparing COVID-19 associated ARDS with ARDS alone, Sihna et al. argue that IL-6 and other cytokines are elevated in COVID-19 associated ARDS, but their levels are much lower than in ARDS, suggesting that COVID-19-related "cytokine storm" may therefore not have an important role as a cause of ARDS (Sinha et al., 2020) . Gerstein et al. reported that in the case of diabetic patients, hypoglycemia is not only the risk factor for cardiovascular and total mortality, but could also be a trigger mechanism for the "cytokine storm" during COVID-19 (Action to Control Cardiovascular Risk in Diabetes Study Group et al., 2008) . In COVID-19 pneumonia, pulmonary thrombosis is common and occurs in two forms: proximal pulmonary embolism and/or distal thrombosis. The main factor involved in J o u r n a l P r e -p r o o f thrombosis appears to be endothelial cell activation (Price et al., 2020) . The presence of viral inclusion bodies has been identified in endothelial cells in several organs from the lungs to the gastrointestinal tract (Varga et al., 2020) . Immune dysregulation can be initiated by pyroptosis, and is a pro-inflammatory form of apoptosis originally described in macrophages (Cookson and Brennan, 2001) . It occurs in severe forms of COVID-19, where it is characterized by rapid viral replication and it leads to the massive release of inflammatory mediators (Li et al., 2020) . In patients with COVID-19, the high rate of pulmonary thrombosis may be the result of three processes: firstly, endothelial inflammation, which leads to in situ thrombosis and microvascular thrombosis; secondly, changes in pulmonary blood flow in response to the parenchymal process, and finally, the classic and very frequent transition from deep vein thrombosis (DVT) to the pulmonary embolism (PE) complication (Klok et al., 2020) . Hypoxic preconditioning (HPC), first described by Lu in 1963 as "a kind of induced tolerance of tissue-cells to hypoxia" refers to exposure of organisms, organs, tissues or cells to a non-injurious, repetitive mild or moderate hypoxic episodes, resulting in increased tolerance and cell protection against subsequent severe hypoxia exposures and other stresses (Lu, 1963) . This preconditioning mechanism has the role of a "warning" signal, which allows the brain and the rest of the organism to prepare for the likely occurence of more harmful conditions (Rybnikova and Samoilov, 2015) . Several phases for hypoxic preconditioning have been described: initiation of hypoxic tolerance in order to promote the immediate adaptation to hypoxia (first phase, during the first few minutes after exposure), followed by the induction of long-term hypoxic tolerance (second phase) and the expression of hypoxic tolerance (third phase, appears after at least 24 h), when pro-adaptive genes are activated. The adaptative effects of normobaric hypoxia preconditioning lasts approximately 72 hours (Rybnikova and Samoilov, 2015) . In order to evaluate the hypoxic status of cells in vitro, cobalt chloride (CoCl2) solution was used as a chemical inducer of hypoxia. Additionally, modular incubator chambers can be used (Wu and Yotnda, 2011) . In vivo, HPC was studied mostly using 8-13% normobaric hypoxia which was achieved by placing animals in a hypoxic chamber. The inhalation of a hypoxic gas mixture through a mask is a non-invasive method suitable for clinical uses of HPC (Rybnikova and Samoilov, 2015) . Targeting HIF-1 for organ protection could be achieved in various ways. Among these, pharmacologic activation of HIF by inhibitors of prolyl hydroxylases (PHDs) was of interest, dimethyloxaloylglycine (DMOG) being reported as a competitive inhibitor against 2-oxoglutarate oxygenases, including PHDs. Others approaches are the direct supplementation of HIF downstream target molecules (such as erythropoietin (EPO) or adenosine receptor agonists) or preconditioning by sevoflurane, an inhalation anesthesic (Lee et al., 2019) . Yao et al. showed that preconditioning with cobalt chloride or desferrioxamine, agents known to increase the stability of HIF-1α, induced J o u r n a l P r e -p r o o f neuroprotective effects in inflammatory disorders of the central nervous system (Yao et al., 2008) . Adenosine signaling is associated with cellular distress conditions and is considered a safety signal in myocardial, hepatic and renal ischemia reperfusion injury. Hypoxia promotes the hydrolysis of the pro-inflammatory ATP released by PMN and injured tissues to adenosine, through the HIF-1 transcriptional activation of endothelial surface proteins CD39 and CD73, that convert ATP to AMP, and respectively AMP to adenosine (Eltzschig et al., 2006; Grenz et al., 2011) . Thiel et al. proved in a acute inflammatory lung injury mouse model that oxygenation inhibits the hypoxia-induced tissue protective ADORA2B signaling pathway and leads to large increases in the mice mortality rate (Thiel et al., 2005) . Hypoxiainduced HIF 1α stabilization activates the extracellular adenosine signaling pathway through the transcriptional activation of ADORA2B, one of the four adenosine receptors. This pathway is part of an endogenous feedback loop that dampens hypoxia-induced inflammation and promotes ischemia tolerance and tissue repair (Poth et al., 2013) . HPC is an effective strategy in several cardiovascular, metabolic, neurological and ventilation respiratory diseases. Daily sessions of intermittent patient exposures to moderate hypoxia interspaced with normoxia seem to be the most promising HPC strategy to develop hypoxia tolerance (Verges et al., 2015) . To date, scientific reports demonstrated that SARS-CoV-2 infection in humans is associated with a large spectrum of clinical respiratory syndromes (Ackermann et al., 2020) . Scientific studies (see below) demonstrate that HPC is related with a variety of biological processes including angiogenesis/vascularization, inflammation, tissue repair and J o u r n a l P r e -p r o o f regeneration, supporting the protective effects of HPC. Considering these effects, a question occurs: could HPC be an effective strategy in COVID-19 therapy? A variety of angiogenic mediators including vascular endothelial growth factor (VEGF), platelet-derived growth factor B (PDGFB), placental growth factor (PGF), angiopoietins 1 and 2, matrix metalloproteinases (MMPs) 2 and 9, plasminogen-activator inhibitor-1, stromal derived factor 1 (SDF1) and stem cell factor (SCF) are induced by HIF-1α activation (Hadjipanayi and Schilling, 2013) . In a study from 2008, Kubo et al. showed that hypoxic preconditioning (culture under 2% O2 for 24 h) activates stress resistance mechanisms in transplanted peripheral blood mononuclear cells (PBMNCs), which increases their survival and angiogenesis induction (Kubo et al., 2008) . In other report from 2013, Li et al. demonstrated that hypoxic preconditioning induces angiogenesis by increasing VEGF and CD31 expression in the ischemic tissue after acute cerebral infarction, thereby protecting brain tissues against ischemic injury (Li et al., 2013) . Furthermore, using a rat model a myocardial infarction, Sasaki et al. showed that hypoxic preconditioning induces myocardial angiogenesis and increases capillary/arteriolar density and blood flow. During hypoxic adaptation, the expression of VEGF was also increased and endothelial apoptosis was decreased (Sasaki et al., 2002) . In myocardial ischemia, potential drug candidates that target the HIF-dependent HPC signaling pathway include: HIF activators, catalysts of extracellular ATP, ADP and AMP hydrolysis to adenosine (nucleotidases and apyrase), ADORA2B agonists and circadian rhythm protein PER2 stabilization promoters . The PER2 promotes the myocardial adaptation to ischemia, while exposure to HPC increases the PER2 protein J o u r n a l P r e -p r o o f synthesis and reduces its proteasomal breakdown through a ADORA2B-dependent mechanism (Eckle et al., 2012) . HPC increases and mobilizes the circulating progenitor cells in a human in a heart injury model. HPC increases the cardiac levels of SDF-1 and VEGF in acute myocardial infarct via EPO signaling, being involved in cardioprotection (Lin et al., 2008) . HIF triggers increases of EPO production and iron uptake, as well as promotes erythroid progenitor maturation and proliferation. All these hypoxia-induced responses increase erythropoiesis. A proposed therapeutic approach for the treatment of anemia induced by insuficient EPO synthesis is the pharmacological targeting of the HIF pathway (Haase, 2010) . Interestingly, hydroxylase inhibitors have been identified as potential drugs to treat anemia, considering their capacity to increased hemoglobin and thus the oxygen-carrying capacity of blood (Huang et al., 2018) . Moreover, hydroxylase inhibitor Vadadustat is in clinical trials for the treatment of ARDS in COVID-19 patients (ClinicalTrials.gov Identifier: NCT04478071) (Bobrow, 2020) . The protective role of hypoxia preconditioning on the brain and the heart exposed to ischemic injury is well established. A similar beneficial effect has been observed on pulmonary function of lung surgery patients (Zhang et al., 2019) . Hypoxia preconditioning promotes survival and decreases apoptosis of pulmonary endothelial cell through a TLR4 based inhibitory mechanism (Ali et al., 2013) . In addition, HIF-1α downregulates ACE2 expression in pulmonary artery smooth muscle cells via angiotensin II production (Zhang et al., 2009 ). Hypoxia reduces inflammation in alveolar epithelial cells through a HIF signaling pathway and HIF activators have shown promising preclinical results in lung injury models. The uptake of HIF activators via the inhaled route has been proposed as a way to reduce their J o u r n a l P r e -p r o o f systemic effects and increase their concentration in the targeted cells (Vohwinkel et al., 2015) . The extracellular adenosine re-uptake through the pulmonary adenosine transporters ENT1 and ENT2 is a crosstalk pathway essential in adenosine-dependent ADORA2B signaling modulation and in lung protection during acute lung injury (ALI). During ALI, the expression of Ent1 and Ent2 is lower, which increases ADORA2B signaling, while Ent1 and Ent2 gene deletion or treatment with an inhibitor (dipyridamole) provides lung protection . disease, which activates the ADORA2B signaling pathway described above, thus protecting the mucosal barrier. A stronger intestinal epithelium protection can be achieved by Ent2 gene deletion or treatment with ENT2 inhibitors (dipyridamole or soluflazine) (Aherne et al., 2018) . HPC dampens systemic inflammation caused by LPS in both mice and humans by activating ADORA2B signaling through increased extracellular adenosine levels, which in turn increases the release of anti inflammatory cytokine IL-10 (Kiers et al., 2018) . Mesenchymal stem cells (MSCs) exposed to hypoxic preconditioning show an enhanced capacity to repair the myocardium after infraction through increases in angiogenesis, vascularization and paracrine signaling, as well as reductions of apoptosis in myocardium (Hu et al., 2008) . Furthermore, enhanced angiogenesis was observed after transplantation of hypoxia preconditioned bone marrow MSCs in rats with cerebral ischemia. These findings suggest that hypoxia-preconditioned transplanted cells exhibit a regenerative capacity and possess therapeutic potential concerning ischemic stroke treatment (Wei et al., 2012) . On the other hand, it has been shown that hypoxic preconditioning of hMSCs improves their osteogenic differentiation (Volkmer et al., 2010) . Novel therapeutic approaches which use stem cells and the extracellular vesicles (EVs) secreted by them could reduce COVID-19-induced inflammation and regenerate the damaged areas of the lung. The resulting therapeutic agents may also be added to established COVID-19 therapeutic protocols (Gupta et al., 2020) . MSCs are a powerful immunomodulatory and anti-inflammatory agent, known to normalize the function of immune systems affected by COVID-19 (Chrzanowski et al., 2020) . MSCs in COVID-19 increase the number of lymphocytes and regulatory dendritic cells, thus increasing their own antiviral protection, reduce the level of C-reactive protein and TNF-α, and increase the level of anti-inflammatory protein IL-10 ( Leng et al., 2020, p. 19 ). The immunomodulatory and regenerative potential of MSCs and their secreted EVs can be clinically useful against the COVID-19-induced "cytokine storm". There are several clinical trials on the therapeutic potential of MSCs against COVID-19. Most of them test MSC-derived exosomes that are administered either intravenously or via inhalation route (Chrzanowski et al., 2020) . There are currently more than 160 different vaccine candidates against SARS-CoV-2 in development (Joszt, 2020) . The anti-COVID-19 vaccine projects of various companies are broadly based on three strategies: using DNA or RNA, weakened virus known as virus-like particles, and targeting viral proteins such as the S protein (Draft landscape of COVID-19 candidate vaccines, 2020). Each of these strategies has therapeutic or economic advantages, but with also some disadvantages (Amanat and Krammer, 2020) . Given the current lack of an effective COVID-19 vaccine, moderate and intermittent hypoxic preconditioning using an oxygen mask connected to a nitrogen cylinder is an easy way to set the organism to face the J o u r n a l P r e -p r o o f effects of various infectious lung diseases. Thus, we propose the use of daily moderate and intermittent hypoxic preconditioning under oxygen status monitoring on the risk group represented by healthy contacts of COVID-19 patients, for the entire SARS-CoV-2 incubation period of (14 days). This is a non-invasive method that could be applied in medical clinics by the health personnel, including medical assistants. On the whole, both vaccination and HPC aim for disease prevention, and not necessary for protection against infection. In the absence of a vaccine, many COVID-19 alternative treatments have been tried, including novel and repurposed drug treatments and the use of plasma from cured patients (S.-J. . Given the lack of any commercially vaccine against COVID-19, HPC should be considered a hormetic approach with very likely beneficial outcomes against a future severe exposure to the same stressor like SARS-CoV-2 without significant side effects. Moreover, our proposed therapeutic approach could be used as a tool of adaptive response monitoring of each individual in hypoxemic condition in order to create a specific profile of high risk developing severe ARDS after infection with SARS-Cov-2. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The publishing fee of this paper has been kindly covered by the Romanian Society of Telemicroscopy. 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