INTRODUCTION

In the majority of cases, the novel coronavirus SARS-CoV-2 causes respiratory disease requiring no special medical intervention, but up to 20% of COVID-19 patients require hospitalization [1]. Severe COVID-19 infection triggers imbalanced and uncontrolled cytokine response (called cytokine storm), exuberant endothelial inflammatory reactions, and vascular thrombosis. These and probably other, yet unknown factors may lead to the development of acute respiratory distress syndrome (ARDS), a major cause of death of the COVID-19 patients [90] demonstrated that pulmonary angiogenesis during COVID-19 was increased by 2.7-fold in comparison with the one in patients died from consequences of the influenza virus A infection. It was suggested that the mechanism behind this phenomenon was based on activated intussusceptive angiogenesis typical to normal development, wound healing, and diverse pathologies [91]. During COVID-19, angiogenesis seems to result from the endothelium damage and hypoxia within the lung injury foci. Elevated ACE2 levels may be related to enhanced angiogenesis or its compensatory expression occurring after virus-mediated blockade of its enzymatic activity, which awaits further investigation (hereinafter the data available in open access on July 20, 2020 are presented). It should be noted that the direct virus-related endothelium damage is a crucial component in pathogenesis of the influenza virus infection as well [92], but intensity of this damage is profoundly lower and it rarely caused severe consequences in comparison with the infection caused by SARS-CoV-2.

Interaction of the lung capillary endothelium with SARS-CoV-2 could occur at early infection stage presuming that potentially it might exit into the bloodstream without disrupting alveolar epithelial cells in close proximity to the capillary endothelium. In the later stages of infection, massive release of the viruses to the blood flow from the large number of destroyed alveolar cells could cause infection of endothelial cells in other vessels. Even without invading sensitive cells, virus may induce some endothelial response by binding to ACE2 and suppressing its proteolytic activity. ACE2 (zinc metalloprotease) cleaves peptide hormone angiotensin-II (ATII), which exerts multiple functions including inducing vasoconstriction causing high blood pressure. Peptides formed due to ATII cleavage may stimulate signaling counteracting ATII. While ACE2 activity declines after binding to viral S-protein, ATII level may be markedly elevated in pulmonary capillaries. Local accumulation of ATII in the lungs was experimentally demonstrated in the bleomycin-induced pulmonary fibrosis model [93]. In this model alveolar epithelial cells and pulmonary myofibroblasts were the main sources of ATII. Systemically elevated ATII in the blood flow during COVID19 seems highly unlikely because high viremia has not been observed even in the severe disease forms, whereas ACE2 is an ubiquitous protein. Up to now, the elevated serum ATII level during COVID-19 infection was reported only in a single study [94].

AT1R serves as a major ATII receptor that activates multilayered signaling in endothelial cells including MAP-kinase axis, protein kinase C, as well as transcription factor NF-κB resulting in activation of NOX2, expression of cytokines, adhesion molecules, and cyclooxygenase 2 (COX2) [95]. Endothelial NOX2 acts as a major source of ROS production, which turned out to be required for AT1R downstream signaling [96]. It was found by Dikalov et al. [97, 98] that stimulation of ATII resulted in the elevated mtROS production that further enhanced NOX2 activity. Experiments with mitochondrial inhibitors and mitochondria-targeted antioxidants revealed that the reduced mtROS production suppressed AT1R signaling. The question regarding the cause of the increased mtROS production still remains open. Dikalov et al. demonstrated that the ATII-induced signaling was lower in the cyclophilin D (CypD) knockout mice [99]. This mitochondrial protein serves as a regulatory component of the mitochondrial permeability transition pore (mPTP), opening of which may result in the increased mtROS level [100]. ROS produced by NOX2 may be one of the causes for pore opening. Thus, ATII initiates positive feedback loop leading to oxidative stress and endothelial dysfunction (figure).

Figure.
figure 1

Angiotensin II (ATII) interacts with AT1R receptor and induces ROS production via NADPH oxidase (NOX) in endothelial cells triggering mitochondrial oxidative stress and endothelial dysfunction. It is believed that SARS-CoV-2 S-protein binds to ACE2 causing its subsequent local or systemic depletion of this enzyme that cleaves ATII, which, in turn, increased ATII level.

Full size image

ATII-induced endothelial activation may occur cooperatively with pro-inflammatory cytokines. In particular, IL-6 stimulates AT1R expression and ATII-dependent signaling that result in the further enhancement of oxidative stress and endothelial dysfunction [101]. IL6 knockout mice exhibit lower endothelial dysfunction caused by administration of ATII [73].

Furthermore, in the acute lung failure model caused by acid aspiration or with bacterial wall lipopolysaccharide ACE2 knockout mice exhibited significantly more substantial tissue damage, whereas recombinant ACE2 or AT1R inhibitor protected from lung injury [102]. Upregulated expression of adhesion molecules, increased secretion of pro-inflammatory cytokines and chemokines, as well as elevated permeability result in acute inflammatory reaction in endothelium in the case of increase of the normal ATII level. Moreover, it is likely to enhance platelet deposition and release of the von Willebrand blood clotting factor that may account for one of causes of develo** thrombosis [103]. Under normal conditions, impact of ATII on thrombogenesis seems to be insignificant [104], but during COVID-19 such effect could be likely. In particular, the pro-inflammatory cytokines may activate platelets in COVID-19 patients that presumably promotes thrombogenesis [105].

Along with endothelial activation, ATII causes secretion of the pro-inflammatory cytokines in alveolar epithelium [106] and changes in the alveolar fluid clearance associated with inactivation of Na+-channels [107]. Moreover, ATII induces alveolar epithelial-mesenchymal transition that causes enhanced epithelial permeability and pulmonary edema [108]. Finally, high ATII levels trigger epithelial cell apoptosis [109].

It should be noted that the hypothesized role of ATII in COVID-19 pathogenesis has been proposed repeatedly (e.g., see [110]), but, however, has not been confirmed experimentally. Drugs blocking ATII production or AT1R signaling are commonly used in treating blood hypertension. Large-scale study conducted by the New York University (NYU) Grossman School of Medicine [111] revealed that taking these drugs does not affect likelihood of infection or risk of severe COVID-19. Using recombinant soluble ACE2 in vitro substantially lowered infection with SARS-CoV-2 owing to competition with the natural ACE2 for virus binding [112]. It can be suggested that such protein would lower ATII level and prevent develo** COVID-19 infection.

At present, no experimental data allowing to assess effects of infection of endothelial cells with SARS-CoV-2 have been obtained. Studies examining interactions between the SARS-CoV-1 and epithelial cells started in 2004 [113] were put on hold. No this type of studies were conducted for the MERS coronavirus. Only few studies aimed at investigating impaired endothelial function after infection with influenza A virus have been reported. In particular, it was shown that this virus in murine model lowered the level of endothelial Krüppel-like Factor 2 (KLF2) [114]. This factor restricts inflammatory activation of endothelium, prevents disruption of permeability and development of atherosclerosis [115]. Interestingly, that Nrf2 is one of the targets of KLF2 in endothelium [116], therefore it is likely that its activation explains protective effects of KLF2.

PERSPECTIVES FOR ANTIOXIDANT THERAPY IN COVID-19

Currently, there are multiple trials underway that test antioxidants as therapeutic agents in COVID-19 (https://clinicaltrials.gov/), but no results were available at the time of preparing current review. Nonetheless, antioxidants such NAC have been already included into the clinical protocols for treating moderate and severe COVID-19 [117].

Most notably, use of antioxidants seems reasonable at the stage requiring inhibition of inflammatory reactions during COVID-19. It is expected that such therapy may prevent organ and tissue damage due to cytokine storm and oxidative stress [118, 119].

Furthermore, lowering oxidative stress by antioxidants may result in the decreased viral load. Recent study with peripheral blood monocytes purified from the healthy volunteers demonstrated that SARS-CoV-2 replication was suppressed by NAC and mitochondria-targeted antioxidant MitoQ [48] allowing to assume that the decreased ROS levels prevent HIF1-α activation and subsequent metabolic switch to glycolysis necessary for coronavirus replication. However, this hypothesis requires further investigation.

Furthermore, To et al. [120] proposed another way of using mitochondria-targeted antioxidant MitoTEMPO in the their study investigating its preventive and therapeutic action in murine model of H3N2 influenza virus infection. In particular, they found that intranasally administered MitoTEMPO decreased mouse mortality, virus titer, as well as lowered airway tract inflammation and decreased neutrophil infiltration. Precise antiviral mechanisms exerted by MitoTEMPO remain obscure; it is likely that the decrease of mtROS results in downregulated expression of the cell-cell adherens junctions, which explains decreased immune cell infiltration as well as reduced activity of NLRP3 inflammasome that produces IL-1β. The decline in virus titer could be explained by the elevated amount of antiviral interferon IFN-1β, which, however, was assessed solely at mRNA rather than protein level. It must be mentioned that the use of MitoTEMPO did not compromise adaptive immune response induced by pulmonary dendritic cells and did not affect population of the lung B and T cell involved in humoral and cellular immunity [120].

Potentially, antioxidants may influence thrombogenesis, which is a common and dangerous complication during COVID-19. Cytokine storm may result in the ROS-dependent apoptosis in endothelial cells [121], whereas SkQ1-mediated mtROS decline prevents TNF-induced apoptosis in vitro [65]. Lowering endothelial cell death could also prevent activation of thrombogenesis.

Another approach in the fight against oxidative stress during COVID-19 involves induction of endogenous antioxidant systems. For instance, the transcription factor Nrf2 controls expression of antioxidant and other cell defense systems. Experiments with mice treated with ATII for 14 days demonstrated that Nrf2 activation with the help of tert-butylhydroquinone lowered ROS level as well as decreased microvascular endothelial dysfunction and hypertension [122]. Similar data were also obtained in vitro in small artery-derived endothelial cells by activating Nrf2 with sulforaphane [123]. The use of Nrf2 activators for COVID-19 therapy is discussed in more detail elsewhere [124].

Thus, the use of mitochondria-targeted antioxidants looks as a promising approach to lower oxidative stress and accompanying complications in viral infections. Further experiments with animal models and clinical trials are necessary to reveal therapeutic potential for such approach.