key: cord-0918485-alqa7pxg
authors: Guan, Shou Ping; Raymond Seet, Chee Seong; Kennedy, Brian K.
title: Does eNOS derived nitric oxide protect the young from severe COVID-19 complications?
date: 2020-11-04
journal: Ageing Res Rev
DOI: 10.1016/j.arr.2020.101201
sha: 7fabe5854f0de90ff8bcd9318634879bb1a57256
doc_id: 918485
cord_uid: alqa7pxg

The COVID-19 pandemic poses an imminent threat to humanity, especially to the elderly. The molecular mechanisms underpinning the age-dependent disparity for disease progression is not clear. COVID-19 is both a respiratory and a vascular disease in severe patients. The damage endothelial system provides a good explanation for the various complications seen in COVID-19 patients. These observations lead us to suspect that endothelial cells are a barrier that must be breached before progression to severe disease. Endothelial intracellular defences are largely dependent of the activation of the interferon (IFN) system. Nevertheless, low type I and III IFNs are generally observed in COVID-19 patients suggesting that other intracellular viral defence systems are also activated to protect the young. Intriguingly, Nitric oxide (NO), which is the main intracellular antiviral defence, has been shown to inhibit a wide array of viruses, including SARS-CoV-1. Additionally, the increased risk of death with diseases that have underlying endothelial dysfunction suggest that endothelial NOS-derived nitric oxide could be the main defence mechanism. NO decreases dramatically in the elderly, the hyperglycaemic and the patients with lowlevels of vitamin D. However, eNOS derived NO occurs at low levels, unless it is during inflammation and co-stimulated by bradykinin. Regrettably, the bradykinin-induced vasodilation also progressively declines with age, thereby decreasing anti-viral NO production as well. These changes with age, low bradykinin and NO, may be thefundamental reasons that intracellular innate immunity declines with age leading tomore severe COVID-19 complications.

 Increase risk of COVID-19 death are also highly related to disease with lower vascular Nitric Oxide (NO) level  Vascular viral defence by endothelial nitric oxide synthase (eNOS) derive NO may be the protecting factor for the young  The negative association between the percentage of eNOS wild type haplotype (T-4b-G) and death per million populations of specific country or ethnicity could potentially explain the disparity of COVID-19 related mortality between Asian and non-Asian countries.

COVID-19 is a devastating disease for the elderly, but relatively mild disease for the majority of younger people (Banerjee et al., 2020; Gandhi et al., 2020) . The molecular mechanisms underpinning the agedependent disparity for disease progression has not been determined. However, a careful interrogation of potential protective factor(s) in young individuals may provide a means to treat the older, more vulnerable population. Based on recently published clinical data, we hypothesize that the endothelial viral defence may play a prominent role in mitigating SARS-CoV-2 complications.

COVID-19 is both a respiratory and vascular disease, especially for those with severe symptoms. The layer of endothelial cells that separates blood from tissues forms an anatomic barrier to viruses within the body. Permeable viruses, especially viremic SARS-CoV-2 (with a size of 80-100 nm), must first infect and replicate in capillary endothelial cells (ECs) before infecting the underlying local tissue (Monteil et al., 2020) . Patients who develop rapid clinical deterioration and death from multi-organ failure exhibit widespread endotheliitis, with accompanying cell death and direct viral infection (Ackermann et al., 2020; Varga et al., 2020) . This indicates that the endothelial barrier has been breached, permitting systemic viral invasion. Moreover, mounting evidence suggests the altered vessel barrier integrity and a pro-coagulative state leads to pulmonary EC damage and, in turn, contributes to the initiation and propagation of acute respiratory distress syndrome (ARDS) (Varga et al., 2020) , which is the main cause of death in patients with COVID-19 (Wu and McGoogan, 2020) . High levels of the fibrin breakdown products, D-dimers, due to an EC death-induced clotting cascade (Nachman and Rafii, 2008) are predictive of poor COVID-19 patient outcome. In contrast, patients with mild signs and symptoms may have their infection resolved without thrombotic complications. These observations suggest that the integrity of endothelial cells is critical to prevent the development of severe COVID-19 (Teuwen et al., 2020) .

The host defence against infection, which can be subdivided into systemic and cell-autonomous immunity, plays a critical role in protecting EC integrity. Systemic immunity detects and clears the infection, destroying the pathogens and the infected cells they inhabit. However, spontaneous EC regeneration is a slow and possibly insufficient process in the context of acute infection (Hirase and Node, 2012) . Hence, the ability to counter infection and to evade host cell death at the individual cell level, termed cell-autonomous immunity (Beutler et al., 2006) , is critical to prevent the spread of the virus. In cell autonomous immunity, the invading viruses trigger production of antiviral agents, including type I and III interferons, β-defensins and nitric oxide that lead to rapid virus clearance. However, viruses have also evolved various strategies to evade these antiviral mechanisms. Serum analysis of COVID-19 patients reveals an elevated pro-inflammatory cytokine and chemokine profile without detectable levels of type I and III IFNs in serum of both the China and USA cohorts (Blanco-Melo et al., 2020; Huang et al., 2020) . Nevertheless, in the absent of these type of interferon, host cells could also mount an antiviral response through the nitric oxide (NO) pathway. Mehta et al. (Mehta et al., 2012) showed that in primary fibroblasts lacking interferon genes, NO production leads to expression of interferon stimulated genes (ISG), as well as other cytokines and chemokines to generate a vigorous antiviral response against both DNA and RNA viruses following poly IC stimulation. Endothelial NOS-derived NO is also a physiological vasodilator, an inhibitor of platelet aggregation and is considered to be a main antiviral defence mechanism. Replication of various RNA and DNA viruses, particularly SARS-CoV (Akerstrom et al., 2005) , is inhibited by NO, which may indirectly decrease viral replication and protein production in host cells by directly inactivating or modifying viral replicating proteins (Abdul-Cader et al., 2016) .

In addition, inadequate production of endothelial Nitric Oxide Synthase (eNOS)-derived NO is a major cause of EC dysfunction, which can lead to disruption of vascular integrity. Established risk factors for COVID-19 mortality, which include old age, hypertension and diabetes mellitus are all characterized by pre-existing endothelial dysfunction (Li et al., 2019) . Endothelial dysfunction leads to vasoconstrictive, proinflammatory and pro-coagulant states, with ensuing organ ischemia and tissue oedema (Bonetti et al., 2003) . Therefore, it is not surprising that patients with pre-existing endothelial dysfunction are also at high risk for adverse outcomes in COVID-19. More critically, newly diagnosed diabetes patients, which have uncontrolled hyperglycaemia, are at much higher risk of COVID-19 mortality than diabetes patients who have taken medication to control glucose levels . This finding may be explained by the observation that high glucose is known to decrease endothelial cell NO bioavailability (Hoshiyama et al., 2003) . Similarly, levels of vitamin D are correlated negatively with mortality in a large study of patients in 20 European Countries. For instance, low vitamin D levels in the aging population of Spain, Italy and Switzerland is associated with increasing vulnerability for COVID-19 mortality (Ilie et al., 2020) . While the pathological mechanisms underlying this observation remains poorly understood, the known role of Vitamin D in upregulating eNOS and NO bioavailability may provide a mechanistic explanation and support a primary role for endothelial viral defense during SARS-CoV-2 infection (Andrukhova et al., 2014) .

Among risk factors, old age is the biggest risk factor for mortality for COVID-19. Severe SARS-CoV-2 infections that require hospitalization are rare in young people, and the risk increases dramatically with age (Docherty et al., 2020) . The reason why SARS-CoV-19 has mostly spared children is not clear, but the marked age-dependent decrease of NO bioavailability level (Cernadas et al., 1998; Lauer et al., 2008; Smith et al., 2006; Yoon et al., 2010) with age could be a major underlying factor sensitizing elders to SARS-CoV-2. Particularly, the activity of eNOS (Cernadas et al., 1998) and endotheliumdependent vasodilatation (Delp et al., 2008; Rodriguez-Manas et al., 2009) progressively declines with age. The reduced bioavailability of NO may thus significantly reduce EC autonomous defences in COVID-19 patients of advanced age.

While the beneficial effects of inhaled NO in ARDS (Rossaint et al., 1995) has been demonstrated and various COVID-19 clinical trials to deliver inhale NO are currently being examined, the importance of eNOS in endothelial cells is not generally considered, as eNOS-derived NO is traditionally perceived as transient and at lower levels than NO derived from inducible Nitric Oxide Synthase (iNOS) (Reiss and Komatsu, 1998) . However, recent studies show that eNOS can also produce prolonged and elevated NO in human ECs under inflammatory conditions and bradykinin co-stimulation (Lowry et al., 2013) . Bradykinin is an important pro-inflammatory and vasodilatory peptide that could be locally induced during tissue damage (Dray and Perkins, 1993) . Bradykinin mediates its vascular actions via 2 types of receptors, bradykinin receptor type 1 (BK1R) and type 2 (BK2R) (Siltari et al., 2016) . Bradykinin-stimulated BK2R in cytokine-treated human ECs intensifies the binding of eNOS to calciumcalmodulin (CaM) at basal Ca 2+ levels, hence inducing the iNOS like NO output (Lowry et al., 2013) . Additionally, eNOS-derived NO also inhibits microbial (e.g. Mycobacterium tuberculosis) growth when the ECs represent the site of microbial invasion (Konradt and Hunter, 2018) , suggesting that eNOSderived NO may be sufficient to play a critical role in ECs autonomous immunity. Regrettably, bradykinin-induced vasorelaxation drops progressively with age (Siltari et al., 2016) , a phenomenon related to the reduced B2R expression in the aged vasculature, therefore establishing the link between aging and COVID-19 sensitivity.

Given the critical role of eNOS in the cell autonomous defence against SARS-CoV-2, eNOS polymorphisms may be directly associated with COVID-19 severity. eNOS is encoded by NOS3 gene, a highly polymorphic and well-studied gene (Cooke et al., 2007) . Some of the NOS3 polymorphisms, such as the promoter single nucleotide polymorphisms (SNPs) g.-786T > C (rs2070744), missense Glu298Asp polymorphism within exon 7 (rs1799983), and a variable number of tandem repeats (VNTRs) in intron 4 (4b/4a), are considered functional; because they affect NOS3 expression or NO formation activities (Oliveira-Paula et al., 2016) . As impairment of endogenous NO production is highly implicated in various diseases, many studies have evaluated the clinical implications of NOS3 polymorphisms either individually or in combination (Oliveira-Paula et al., 2016) . Haplotype analysis is often performed as SNPs may affect one another and are often inherited as a unit (Crawford and Nickerson, 2005) .

Although the relevance of individual NOS3 polymorphisms to various diseases have been discovered, the findings are often conflicting (Cooke et al., 2007; Pereira et al., 2007) . These discrepancies may be related to the analysis of single genetic polymorphisms that neglect the interactions between other polymorphisms within NOS3 (Crawford and Nickerson, 2005; Tanus-Santos and Casella-Filho, 2007) . For example, while individual NOS3 polymorphisms did not affect plasma nitrite/nitrate (NOx) levels, the C-4b-Glu haplotype was associated with decreased NOx levels (Metzger et al., 2011; Metzger et al., 2005) , suggesting that haplotype analysis may provide improved predictivity and inform better about the risks of specific genotypes (Crawford and Nickerson, 2005) . In terms of haplotype analysis, the WT haplotype, which combines the wild-type variants for all three polymorphisms (T-4b-Glu) is the most common haplotype found in all populations studied (Luizon et al., 2009; Marroni et al., 2005; Tanus-Santos et al., 2001; Thomas et al., 2013) , and has been least associated with all diseases examined (Kitsios and Zintzaras, 2010) . In fact, it has been generally linked to be a protective phenotype (Kumar et al., 2009) . Nonetheless, there is notable disparity in the distribution of WT haplotypes between different ethnic groups, with the frequency of the WT haplotype was much higher in Asians than in all other ethnic groups (Qi et al., 2020; Tanus-Santos et al., 2001) . These findings could partially explain the lower incidence of cardiovascular diseases in Asians (Tanus-Santos et al., 2001) . While COVID-19 morbidity and mortality are influenced by age, specific pre-existing health conditions and longstanding discriminatory societal and historical factors, the contribution of underlying biological factors to the burden of COVID-19 cannot be dismissed as many countries are witnessing differential health outcomes according to ethnicity. A recent study on the global clinical characteristics and mortality for COVID-19 discovered that hospital mortality was significantly higher in America and Europe than to Asia (Goel et al., 2020) . More strikingly, there was a direct negative correlation between COVID-19 mortality per 100K people when compared to the percentage of NOS3 WT (T-4b-Glu) haplotype based on available data (Figure 1 (Qi et al., 2020) , South Korea (Shim et al., 2010) , India (Nishank et al., 2013 ), Saudi Arabia (Alkharfy et al., 2012 , Columbian (Average of Black and White) (Serrano et al., 2010) , US (White, Black) (Thomas et al., 2013) , Brazil (Sandrim et al., 2006) , Spain (Amoli et al., 2003) .

This finding may explain the disparity in global mortality among Asians and other continents, especially when comparing countries with cases (as of October 2020), such as the US and India, where the death per million is significantly lower in India despite lower access to medical resources among infected individuals. This striking association further strengthens the hypothesis that eNOS levels provide robust defence against COVID-19, and warrants further clinical trials in the development of treatment strategies that enhance endothelial NO levels.

Taken together, all the connecting points strongly suggest that the progression of COVID-19 can be hindered by a strong endothelial cellular defence system through induction of NO. This hypothesis provides a rationale for therapies to increase endothelial NO as a means to treat hospitalized moderateto-severe patients, particularly for older patients that have low NO levels, through drugs that upregulate NO bioavailability, such as nitrite anions, nitric oxide agonist (minoxidil), steroid hormones (dehydroepiandrosterone and estrogen), HMG-CoA reductase inhibitors (statins), resveratrol, and folic acid (Cau et al., 2012) . In order to prove the validity of this hypothesis, further studies and clinical trials are warranted. 

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