MBBS Gemma A Figtree
email: gemma.figtree@sydney.edu.au.

Barbara Casadei

Robert Califf

Filippo Crea

Grant Drummond

Jane E Freedman

Tomasz Guzik

David Harrison

Derek J Hausenloy

Joseph A Hill

James Januzzi

Bronwyn A Kingwell

Carolyn S P Lam

Calum A Macrae

Frank Misselwitz

Tetsuji Miura

Rebecca Ritchie

Maciej Tomaszewski

Joseph C Wu

Junjie Xiao

Faiez Zannad

Dphil

A CALL TO ACTION FOR NEW GLOBAL APPROACHES TO CARDIOVASCULAR DISEASE DRUG SOLUTIONS

Keywords: drug discovery, pharmacotherapy, cardiovascular, organoids, precision medicine

Whilst we continue to wrestle with the immense challenge of implementing equitable access to established evidence-based treatments, there remain substantial gaps in our pharmacotherapy armament for common forms of cardiovascular disease including coronary and peripheral arterial disease, heart failure, hypertension and arrhythmia. We need to continue to invest in the development of new approaches for the discovery and rigorous assessment of new therapies. Currently, the time and cost to progress from lead compound/product identification to the clinic, and the success rate in getting there reduces the incentive for industry to invest, despite the substantial burden of disease and potential size of market. There are tremendous opportunities with improved phenotyping of patients currently batched together in syndromic "buckets". Use of advanced imaging and molecular markers may allow stratification of patients in a manner more aligned to biological mechanisms that can, in turn be targeted by specific approaches developed using high throughput molecular technologies. Unbiased "omic" approaches enhance the possibility of discovering completely new mechanisms in such groups. Furthermore, advances in drug discovery platforms, and models to study efficacy and toxicity more relevant to the human disease are valuable. Reimagining the relationship between discovery, translation and implementation will help reverse the trend away from investment in the cardiovascular space, establishing innovative platforms and approaches across the full spectrum of therapeutic development.

Introduction

Cardiovascular disease is the leading cause of death globally, with an ongoing rapid rise in low and middle income countries [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF][START_REF] Virani Salim | Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association[END_REF] . Initiatives to improve equitable access to and implementation of established effective therapies (e.g. statins) continue to be an international challenge. However, there remain substantial gaps in our pharmacotherapy armament for common forms of cardiovascular disease, including coronary and peripheral arterial disease, heart failure, hypertension and arrhythmia. Collaborative efforts to tackle access and affordability issues need to be combined with initiatives to accelerate new therapeutic discovery and development. Integration of health economic analyses at all stages of the development and trial pipeline, that take into consideration the "payer's" perspective relevant to the different jurisdictions, is a critical factor for successful and equitable implementation. Currently the time and cost to progress from lead compound/product identification to the clinic, and the success rate in getting there reduces the incentive for industry to invest, despite the substantial burden of disease and potential market size. Re-imagining the relationship between discovery, translation and implementation is required to rapidly reverse these trends. This can be achieved by establishing innovative platforms and approaches across the full spectrum of therapeutic development, and re-establishing a thriving and impactful sector capable of discovering and translating new solutions.

Where are we now?

Whilst there have previously been limited specific data on development of cardiovascular drugs, and factors associated with success of new therapies in clinical trials, Hwang and colleagues have provided an excellent quantitative analysis of the field [START_REF] Hwang | Temporal Trends and Factors Associated With Cardiovascular Drug Development, 1990 to 2012[END_REF] . Prior to this, reports had anecdotally suggested that the number of new cardiovascular drugs approved by the US Food and Drug Administration (FDA) had recently declined [START_REF] Fordyce | Cardiovascular drug development: is it dead or just hibernating?[END_REF][START_REF] Darrow | Drug development and FDA approval, 1938-2013[END_REF] , and that there was a contraction in the pool of drugs under development targeting cardiovascular disease [START_REF] Pammolli | The productivity crisis in pharmaceutical R&D[END_REF] . Using the large commercial database of drug development activity (Citeline Pharmaprojects, Informa plc), Hwang et al were able to quantify the rate and ultimate success of translation along the pipeline of over 50,000 products for all diseases, and to compare the rates among disease states, as well as examine factors associated with success. These data confirmed that the number of new cardiovascular drugs entering clinical trials at all stages of development declined between 1990 and 2012 (compared with cancer which increased). Many cardiovascular drugs in development were targeted against modifiable risk factors and previously proven mechanisms addressing these risks including antihypertensive agents, lipid modifying agents, and anticoagulants. However, 50% of cardiovascular drugs entering phase III trials between 1990 and 2012 were categorised as targeting novel biological pathways, with this rate of novelty increasing over the time period. Most clinical trials for cardiovascular drugs were sponsored by large pharmaceutical companies. Smaller companies were observed to be active, but more focussed on rarer forms of cardiovascular disease. In all three phases of study, the proportion of cardiovascular trials sponsored by large pharmaceutical companies compared to small-or medium-sized companies was significantly greater than that for non-cardiovascular trials [START_REF] Hwang | Temporal Trends and Factors Associated With Cardiovascular Drug Development, 1990 to 2012[END_REF] .

At the center of the disparity between cardiology and oncology is the lack of routine clinical access to tissue in cardiac and vascular diseases which has led to disease classifications and thus clinical trials focused to a large extent on phenotypes that aggregate relevant visible properties. These phenotypes, while often linked by final common pathways, typically reflect heterogeneous underlying etiologies and thus responses to intervention. Therefore, efforts in cardiology (and other specialties) to reclassify or stratify diseases based on discrete pathogenetic mechanisms have been proposed as one rational approach to resolving the trend away from cardiovascular therapeutic development. Identification of etiologically homogeneous subgroups of patients (endotypes) within a heterogeneous population is a goal of stratified medicine but remains a challenging. For instance, Type 1 myocardial infarction is a phenotype which is caused by different endotypes needing different forms of therapy [START_REF] Crea | Acute Coronary Syndromes: The Way Forward From Mechanisms to Precision Treatment[END_REF] , which may not be readily defined by current techniques. Similarly, angina is a phenotype which is caused by different endotypes [START_REF] Kaski | Reappraisal of Ischemic Heart Disease[END_REF] , but we have tended to focus only on the endotypes for which we have efficient interventions (i.e. macrovascular obstruction). This, and some of the additional challenges and potential solutions are outlined below and summarised in Table 1.

Challenges

Potential solutions Historic tendency to target biological mechanisms which have empirically been shown to influence overall outcomes, rather than definitive disease mechanisms (necessary and sufficient) identified in a discrete subset. Thus ~50% agents entering phase III clinical trials still target BP, lipids and coagulation 3 , with limited novel therapeutic targets beyond these.

Identification of definitively causal (not association) novel targets and pathways from robust studies of increasingly homogeneous phenotypic subsets through improved use of molecular and advanced imaging and clinical markers. Increasing proportion of CVD drugs entering phase III trials targeting novel causal biologic pathways 3, 9 Limitations in methods that target known signalling pathways involved in disease processes Advances in drug design platforms will accelerate translation of complex mechanistic knowledge to new therapies. Poor translation of safety and efficacy parameters from preclinical models (especially rodents) to humans Development of mechanistically faithful human exvivo cardiac and vascular organoids, and the creation of similarly robust animal models that do not just mimic the human condition, but rather reflect the definitive mechanisms, and the burden of co-morbidities and age. Lack of rigor in some preclinical research (e.g. single centre, not blinding or randomising, no protocol publication, over emphasis of findings) contributes to well-recognised limited reproducibility

Residual risk and "missing" biology

Many medications in cardiovascular disease have been identified through serendipity, for example, none of the major classes of drug treatment for heart failure (renin-angiotensinaldosterone inhibitors, beta blockers, SGLT2 inhibitors), were initially developed for the syndrome. Not surprisingly, drugs discovered in this manner do not appear to address all of the pathophysiologic components of the diseases which they are now used to treat. For example, up to 27% of ST-elevation MI patients do not exhibit traditional modifiable risk factors [START_REF] Vernon | Increasing proportion of ST elevation myocardial infarction patients with coronary atherosclerosis poorly explained by standard modifiable risk factors[END_REF][START_REF] Vernon | ST-Segment-Elevation Myocardial Infarction (STEMI) Patients Without Standard Modifiable Cardiovascular Risk Factors-How Common Are They, and What Are Their Outcomes?[END_REF] , and a substantial proportion of patients with coronary artery disease (CAD) progress rapidly to recurrent acute events despite optimal medical management [START_REF] Schiele | Coronary artery disease: Risk stratification and patient selection for more aggressive secondary prevention[END_REF] . The recent successful trial addressing the residual risk driven by inflammation (CANTOS [START_REF] Ridker | Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease[END_REF] and LoDoCo2 [START_REF] Nidorf | Colchicine in Patients with Chronic Coronary Disease[END_REF] ) are good examples of how cardiovascular risk can be further reduced addressing non traditional risk factors. A major limitation of these trials, however, is that they have included the broad population of patients with cardiovascular disease rather than the "endotype" susceptible to an anti-inflammatory treatment [START_REF] Crea | Addressing Acute Coronary Syndromes: New Challenges and Opportunities After the CANTOS Trial (Canakinumab Anti-inflammatory Thrombosis Outcomes Study)[END_REF] .

There is significant potential to address these gaps through systematic reappraisal of the mechanisms of the constituent disorders, and the application of proven methods of drug discovery to the resultant novel mechanistically robust targets. For quite some time cardiovascular discovery efforts have been dominated by a multitude of signalling pathways, emerging from large-scale profiling technologies, which while they often provide molecular phenotyping [START_REF] Vernon | Utilizing state-of-the-art "omics" technology and bioinformatics to identify new biological mechanisms and biomarkers for coronary artery disease[END_REF] , often lie downstream of the actual causal factors and in many instances may represent compensatory pathways, at least partially explaining the mixed results that have been observed. For example, of the >150 genetic loci reproducibly associated with CAD to date, very few are thought to act via traditional risk factors [START_REF] Deloukas | Large-scale association analysis identifies new risk loci for coronary artery disease[END_REF] . Pipelines focused on discoveries from genome-wide association studies (GWAS) have been employed by a number of the leading Pharma companies for over a decade, but new causal pathways and successful interventions based on such targets have been slow to emerge.

Technology to target novel biology

The technologies to efficiently develop new therapeutics once definitive targets have been identified are maturing and approaches including in silico screening, fragment-based drug discovery and tagged libraries have changed the pace of modern drug discovery [START_REF] Murray | The rise of fragment-based drug discovery[END_REF] . An expanded tool kit of molecular targeting through cell, gene, RNA, peptide, and protein (including antibody) therapies and nanoparticle technology provide further diversity of approaches and enhance the likelihood of success against any target [START_REF] Bubb | New opportunities for targeting redox dysregulation in cardiovascular disease[END_REF][START_REF] Landmesser | From traditional pharmacological towards nucleic acid-based therapies for cardiovascular diseases[END_REF] . RNA and gene therapies offer remarkable specificity and safety profiles with successful 'platform' approaches to therapeutic development, for example, the siRNA Inclisiran (Novartis) represents a powerful approach to sustained inhibition of PCSK9 to reduce LDL-cholesterol. The top 10 (2019) highest growing pharmaceutical companies that are using artificial intelligence (AI) or machine learning for drug discovery have been recently reviewed 22 .

Patients with recurrent events despite best evidence-based care-an opportunity for novel agents

The current unmet need in cardiovascular disease is exemplified by the substantial number of individuals who develop disease or progress despite best practice treatment. Examples include resistant hypertension (estimated to be ~10% of the hypertensive population) and those with advanced atherosclerosis and acute MI, but no elevation in Standard Modifiable cardiovascular Risk Factors ("SMuRF"-less; between 15-30% of ST elevation MI [START_REF] Vernon | Increasing proportion of ST elevation myocardial infarction patients with coronary atherosclerosis poorly explained by standard modifiable risk factors[END_REF][START_REF] Vernon | ST-Segment-Elevation Myocardial Infarction (STEMI) Patients Without Standard Modifiable Cardiovascular Risk Factors-How Common Are They, and What Are Their Outcomes?[END_REF] ). A key goal is to define those individuals at risk of rapid cardiovascular disease progression, in a manner similar to metastatic malignancy, where new agents can be rapidly tested in small sample sizes due to higher event rate. This approach permits elucidation of novel biological mechanisms not currently addressed by existing agents (Figure 1). For instance, recent data suggests that failure of plaque healing might help explain recurrent acute coronary events and might become a new therapeutic target [START_REF] Vergallo | Atherosclerotic Plaque Healing[END_REF] . A consensus definition of those who rapidly progress to have recurrent events for atherosclerosis, heart failure or other disorders, despite best evidence-based therapies may also provide enhanced opportunities to define underlying mechanisms, and to augment the power for targeted therapeutic studies. Similarly, angina or myocardial infarction without obstructive coronary disease, a cause of substantial morbidity with unmet need, might usefully be defined using population level diagnostics for coronary microvascular dysfunction which could then drive rigorous mechanistic studies and improved targeted therapies [START_REF] Camici | Coronary microvascular dysfunction[END_REF] .

Figure 1. Schematic representation and summary of 5 patient groups with unmet need for new therapeutic approaches.

Precision medicine and drug development

Common disease states impacting cardiovascular health, driving morbidity and mortality such as atherosclerosis and heart failure, are multi-factorial, and have been shown to consist of many different sub-groups each presumed to be driven by a distinct predominant mechanism (as yet unknown). An extreme example is the syndrome of heart failure which is divided into subgroups based on systolic function-reduced ejection fraction (HFrEF) versus preserved ejection fraction (HFpEF). Interestingly, HFrEF patients, with widely diverse aetiologies-from prior myocardial infarction, to chronic ischemia, to inflammatory and infiltrative conditions, and through to dilated cardiomyopathy of uncertain aetiology, all appear to gain some benefit from the same neurohormonal agents. In contrast, clinical trials of patients with HFpEF have been consistently neutral [START_REF] Yoon | Heart failure with preserved ejection fraction: present status and future directions[END_REF] . This may partly relate to the even more broad spectrum of phenotype cluster, which not only includes individuals with bona fide physiologic abnormalities of relaxation e.g. cardiac amyloidosis and restrictive cardiomyopathy, but also an amalgam of conditions where non-cardiac comorbidities and abnormalities contribute, and the only cardiac abnormalities may be changes in load dependent indices of diastolic physiology which may represent a range of disorders from deconditioning to poorly treated hypertension [START_REF] Shah | Precision Medicine for Heart Failure with Preserved Ejection Fraction: An Overview[END_REF][START_REF] Dunlay | Epidemiology of heart failure with preserved ejection fraction[END_REF] . A similarly complex spectrum of "phenotypes" and aetiologies, regarding individual patterns of response to classic risk factors, likely exists for atherosclerosis [START_REF] Kott | Biobanking for discovery of novel cardiovascular biomarkers using imaging-quantified disease burden: protocol for the longitudinal, prospective, BioHEART-CT cohort study[END_REF] . Better stratification by underlying mechanistic markers will be required to make the next advances in the prevention and treatment of these conditions.

Current clinical trials, and subsequently guidelines, of necessity tend to "bundle" patients together based on the aggregated syndromes referred to above. Whilst the evidence-based treatment likely acts on common parts of the pathway, and some pharmaceutical companies would prefer to design validation phases to provide as "wide" a market as possible, this may hinder larger leaps relevant to smaller more etiologically pure sub-groups. Advances in disease stratification tools (including molecular and advanced imaging technologies) to identify more specific patient populations will help to unravel new mechanisms in these subgroups, and lead to better targeting of new therapies. Ultimately, these same mechanisms will also be useful to identify potential novel endpoints that will also accelerate subsequent focused clinical trials. In other words, time has perhaps come to give more emphasis to "splitting" rather than "lumping".

AI and machine learning approaches are being used to better cluster patient populations and derive more tightly circumscribed patient cohorts. This patient segmentation may derive patient endotypes, but this will likely require a systematic improvement in the information content of phenotypic assessment. Both unsupervised and supervised machine learning approaches are currently being used to better characterize patients with heart failure and those who have had a stroke. Among these efforts are electronic health record-based explorations of several million patients with longitudinal follow-up data (up to 7 years) in a collaboration with the Broad Institute at Harvard and MIT, Boston, MA, US, as well as with Sensyne Health in Oxford, UK. The American Heart Association have also invested substantially in the Precision Medicine Initiative. The resulting datasets offer the opportunity to train models against discrete outcomes and then validate these in prospectively collected data from Randomized Controlled Trials (RCT). Such approaches aim to improve inclusion or exclusion of patients in future clinical trials targeting more distinctly stratified patient cohorts, but ultimately definitive new biology will be required to define new targets.

A more immediate application of genomics to drug development, is the relatively simpler application of pharmacogenomics, where variants at a specific locus are known to alter the metabolism or response to therapeutic agents. There has been mixed support for such an approach from Pharma yet targeting drugs to specific mechanisms in individuals might streamline development pathways with smaller trials and ultimately lead to higher efficacy and safety as well as better outcomes for patients. In the case of clopidogrel, warfarin and statins, drugs already well-established, the literature has become sufficiently strong that guidelines are now available to help implement the use of genetic information to guide treatment with these therapies [START_REF] Johnson | Pharmacogenetics and cardiovascular disease-implications for personalized medicine[END_REF] . The cost effectiveness of implementing these insights has been difficult to prove, and so the prospective development and validation of new therapies for patients with specific genotypes has not been broadly undertaken.

Advances in in vitro human models of cardiovascular disease

Pharmacological studies have long been a centrepiece of nonclinical drug evaluation prior to in vivo studies. Well-defined cellular models enabling investigators to screen chemical compound libraries and drug candidates have then been selected for secondary validation screens. Perhaps more than other organs, there is a need for in vitro models that better represent the human heart to understand cellular mechanisms and to test new therapies [START_REF] Ebert | Induced pluripotent stem cells as a disease modeling and drug screening platform[END_REF] . Several teams around the world have made recent progress on this front by moving from 2D monolayer cultures to 3D spheroids, organoids, or microphysiological systems. This includes both primary cardiac myocytes, or induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), along with co-culturing approaches which incorporate endothelial cells [START_REF] Sayed | Clinical trial in a dish using iPSCs shows lovastatin improves endothelial dysfunction and cellular cross-talk in LMNA cardiomyopathy[END_REF] , and cardiac fibroblasts [START_REF] Zhang | Generation of Quiescent Cardiac Fibroblasts From Human Induced Pluripotent Stem Cells for In Vitro Modeling of Cardiac Fibrosis[END_REF] . Such approaches not only enhance the modelling of human heart behaviour, but also prolong the survival of human cardiac myocytes in culture [START_REF] Polonchuk | Cardiac spheroids as promising in vitro models to study the human heart microenvironment[END_REF] . For example, Mei and colleagues have established a model of myocardial infarction in human cardiac organoids that utilises both hypoxia and noradrenaline and recapitulates pathological metabolic, fibrosis, and calcium handling changes at the transcriptomic, structural and functional level [START_REF] Sayed | Clinical trial in a dish using iPSCs shows lovastatin improves endothelial dysfunction and cellular cross-talk in LMNA cardiomyopathy[END_REF] . Hudson and team have developed a bioengineered human cardiac organoid platform which provides functional contractile tissue with biological properties similar to native heart tissue. Moreover, the team have applied this model to screen small molecules with pro-regenerative potential, demonstrating that whilst some small molecules were proregenerative, they had the unintended side effect of decreasing contractility [START_REF] Zhang | Generation of Quiescent Cardiac Fibroblasts From Human Induced Pluripotent Stem Cells for In Vitro Modeling of Cardiac Fibrosis[END_REF] . Similarly, Takeda et al., have demonstrated the potential of 3D cardiac tissue derived from human iPSC-CMs, to screen for drug-induced cardiotoxicity [START_REF] Takeda | Development of In Vitro Drug-Induced Cardiotoxicity Assay by Using Three-Dimensional Cardiac Tissues Derived from Human Induced Pluripotent Stem Cells[END_REF] . Such approaches have the potential to support early drug screening and tackle the biggest gaps in our drug armament, but it will be vital to improve the uniformity of the organoids at the scale necessary for empiric screening, and proof of translatability remains a challenge for all preclinical models in the cardiovascular space. If successful, this will be a major step forward in overcoming the limitations related to our minimal access to the tissues and cells at the centre of the disease.

In vivo translational models

Robust demonstration of both proof-of-mechanism and efficacy in animal disease models is a critical step in drug development that must be achieved prior to progression to human trials. The cardiovascular field is littered with numerous examples of therapies that show great promise in preclinical studies yet fail during the clinical trial phase. A major challenge for cardiovascular drug development, that is shared by other disease groups, is the failure of many small animal (usually rodent) models to fully reproduce human diseases. For example, rodents are particularly resistant to the development of atherosclerosis, and even more so to the development of unstable plaques. While the reasons for this are not fully understood, they are at least partly related to fundamental species differences in hemodynamics, lifespan, lipid metabolism (e.g. absence of the cholesteryl ester transfer (CETP) gene in rodents) [START_REF] Ha | Differences in plasma cholesteryl ester transfer activity in sixteen vertebrate species[END_REF] and immune function [START_REF] Zschaler | Differences in innate immune response between man and mouse[END_REF] . Similar difficulties in adequately replicating disease parameters in animal models for other human cardiovascular pathologies, including cardiac arrhythmias (where species differences in cardiac electro-mechanical coupling confound model development [START_REF] Milani-Nejad | Small and large animal models in cardiac contraction research: advantages and disadvantages[END_REF] ), HFpEF, and resistant hypertension, have plagued translation of discovery research into the clinic. Typically, cardiovascular disease models are agnostic to the actual disease mechanisms in humans, not least because there is insufficient information available regarding the biology in humans as a result of the logistical challenges of obtaining human cardiac and vascular disease tissues.

Practices that are routine in clinical research such as a priori sample size and power calculations, randomisation, blinding, and the use of appropriate statistical analyses, were frequently overlooked in preclinical studies, particularly in academic research. Other factors such as clearly defined inclusion and exclusion criteria, littermate controls, full disclosure of all collected data, and confirmation of findings across multiple labs, have historically often been lacking in academia [START_REF] Drucker | Never Waste a Good Crisis: Confronting Reproducibility in Translational Research[END_REF][START_REF] Hausenloy | Translating Cardioprotection for Patient Benefit: The EU-CARDIOPROTECTION COST Action[END_REF][START_REF] Lindsey | Guidelines for measuring cardiac physiology in mice[END_REF] . Many cardiovascular models are 'accelerated', for example employing animal models in which the disease phenotype of e.g. atherosclerosis or heart failure develops over weeks rather than months to years, and/or utilising young animals despite the fact that in humans most cardiovascular diseases present during middle age or beyond. Similarly, many therapies are tested in remarkably few genotypes before clinical use, so that relatively rare adverse events may not be detected. Likewise, cardiovascular morbidities seldom present in isolation from other co-morbidities, yet few studies focus on animal models that combine multiple diseases (e.g. superimposing comorbidities such as obesity or hypertension onto the model of interest). Finally, the overwhelming majority of animal studies only use males and thus likely fail to model key disease mechanisms present in females, failing to represent half of the patients for whom these therapies are intended in the real world.

How can the field be improved? First and foremost is the need for rigorous mechanistically faithful models for the indication of interest. Attention to those models which have proven translatability for safety and efficacy in previous drug development programs would also be useful [START_REF] Hausenloy | Translating Cardioprotection for Patient Benefit: The EU-CARDIOPROTECTION COST Action[END_REF] . Where there are no mechanistic models, efforts are likely best focused on human studies to define causal mechanisms. In many instances there is also a need for large animal models, as a bridge between rodent studies and human trials, but here too the need for mechanistic representation cannot be overstated. Finally, closer attention must be paid to best practice in experimental design. In this regard, guidelines such as ARRIVE (Animal Research: Reporting of In Vivo Experiments) [START_REF] Du Sert | The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research[END_REF] and STAIR (Stroke Therapy Academic Industry Roundtable) [START_REF] Savitz | Stroke Treatment Academic Industry Roundtable X: Brain Cytoprotection Therapies in the Reperfusion Era[END_REF] , modelled after the CONSORT (Consolidated Standards for Reporting Trials) statement for clinical research, provide an excellent roadmap. As such, a template flow diagram for reporting animal use and analysis in preclinical studies has been developed [START_REF] Drucker | Never Waste a Good Crisis: Confronting Reproducibility in Translational Research[END_REF] , the Consolidated Standards of Animal Experiment ReporTing (CONSAERT) template.

Choosing the right models and the right endpoints, as well as empirically defining the extent to which the modeled biology and response to intervention translate into humans, should be a quantitative metric for filing with regulatory agencies to move toward clinical studies. This requirement would also enable such data to be made public in a protected manner, thus enabling progressive optimization of the drug development process.

Transition to a staged approach for approval of therapies based on clinically and mechanistically relevant measures of benefit and safety

The high bar that the cardiovascular field has set, with primary endpoints focussed on mortality in sharp contrast with oncology in which surrogate end points are frequently accepted, creates an unfortunate backlash of disincentivising investment due to high costs, and high risk. Whilst being careful not to lower the ultimate bar, collaborative effort to identify reproducible markers of benefit and safety that could be tested in a staged approach, may be required to ensure efficiency of the cycle, and ongoing investment in the development of cardiovascular drugs. A focus on what is important to the patient, as well as on the biology underlying a more precise disease taxonomy, will be important.

In 2015 the FDA approved alirocumab and evolocumab, monoclonal antibodies to PCSK9, for high risk patients, prior to the large cardiovascular outcome trials being completed 3 due to their benefit on lipid profiles in those who were already maximally treated. This is not dissimilar to the pathway that the diabetic field has taken as required by FDA regarding glucose-lowering treatments, and the oncology field with cancer "shrinkage". In each instance there is a substantial tension between such limited approval and the need for demonstration of definitive benefit and avoidance of harm for patients in terms of morbidity and mortality. Innovation in patient-reported or functional endpoints can be accelerated with the arrival of wearables and other new technologies. There is some potential for advances in non-invasive imaging of the myocardium (e.g. post-contrast myocardial T1 mapping for cardiac fibrosis [START_REF] Vo | Society for Cardiovascular Magnetic Resonance (SCMR) expert consensus for CMR imaging endpoints in clinical research: part I -analytical validation and clinical qualification[END_REF] ) and atherosclerosis (e.g. serial CT coronary angiography and plaque characterisation [START_REF] Vaidya | Colchicine Therapy and Plaque Stabilization in Patients With Acute Coronary Syndrome: A CT Coronary Angiography Study[END_REF] ) to enhance the power of early phase trials for relevant drugs, but it will be vital to improve the rigorous mapping of such metrics onto meaningful patient outcomes. The "hard" endpoints required for new agents for management of myocardial infarction, heart failure, or most primary prevention drugs is a consequence of prior adverse outcomes when such putative surrogates were employed. Improving our approaches to defining and testing new endpoints will be a central requirement to advance cardiovascular drug discovery. The advent of real-world tools and large-scale analytics may aid this process, but here again increased information content will likely be necessary to escape the constraints of existing disease nosology and the resultant decades of downstream selection bias. Regulatory agencies have strong track records responding to patient-centred outcomes, and the cardiology community is actively increasing the use of such metrics and novel trial designs to transform the approaches to translational science in the field. A collaborative effort working with regulatory agencies to develop a staged approach may allow for acceleration of efficacious therapies, reducing time and cost, but not rigor and safety.

There is increasing appreciation of the importance of early engagement with patients and the community in regard to endpoints that are most relevant and important to them. In this regard, it is critical that the cardiovascular sector is proactive in consumer engagement in the design of studies, and in developing innovative measurement of patient-reported outcomes and experiences. Such patient-reported outcomes can be captured electronically or in person through formats ranging from questionnaires to wearable devices and are used to provide a quantifiable record of the patient's lived experience of a disease. The inclusion of such measures as trial endpoints allows for a more comprehensive assessment of the burden of disease and the impact of an intervention [START_REF] Calvert | Guidelines for Inclusion of Patient-Reported Outcomes in Clinical Trial Protocols: The SPIRIT-PRO Extension[END_REF] . However, for such endpoints to be accepted by regulatory bodies to inform decisions regarding the value and efficacy of the intervention, we need to ensure rigor and reproducibility.

Innovative clinical trial design

One consequence of disease heterogeneity is diminution of the average therapeutic effect.

Because of these modest effect sizes, the large-scale, long term follow-up phase III clinical trials needed to demonstrate the effect of interventions on the essential outcomes of CV disease ("hard" cardiovascular endpoints of mortality or combined Major Adverse Cardiovascular Events (MACE)) are expensive and require time for follow-up. Consensusbuilding between pharmaceutical manufacturers and regulatory bodies, and the inclusion of multiple other stakeholders, to ensure meaningful problems are addressed directly is a key factor for consideration [START_REF] Marquis-Gravel | Improving the Design of Future PCI Trials for Stable Coronary Artery Disease: JACC State-of-the-Art Review[END_REF] . Diversity of the trial population, specifically with appropriate balance of women and different ethnic groups, could also be significantly improved [START_REF] Marquis-Gravel | Improving the Design of Future PCI Trials for Stable Coronary Artery Disease: JACC State-of-the-Art Review[END_REF] . At the same time, improved stratification based on precise phenotypes will improve power and success. The estimated cost of a Phase III clinical trial for primary prevention in cardiovascular disease is between $USD250-450 million; approximately 20-times higher than the median estimated cost of Phase III clinical trials overall. Thus, improving the efficiency of cardiovascular trials would be an important step to reinvigorate interest, and to accelerate new drug development and translation at all phases of the pipeline.

The advent of electronic health records and potential for routinely collected medical data to populate virtual registries for heart attacks, heart failure and arrhythmia as examples, are one of a number of opportunities to enhance trial design efficiency and lower costs. Utilization of routinely collected electronic health data will allow self-population of electronic case report form entries to enhance efficiencies of registries and randomized clinical trials. It may also enable the use of additional synthetic or semi-synthetic control arms by matching verum cases with placebo cases from existing patients with the same demographics and baseline characteristics and with an appropriately long enough longitudinal follow-up. Furthermore, the efficient use of electronic medical record data may enhance the integration of the health system with the research and development (R&D) sector, increasing the proportion of patients with specific conditions participating in trials.

The impact of innovation in clinical studies of novel therapies is evident in Astra Zeneca's testing of Farxiga which has been granted Fast Track Designation in the US for heart failure following acute myocardial infarction leveraging a registry-based trial design [START_REF]acute myocardial infarction leveraging an innovative registry-based trial design[END_REF] . Inclisiran (Novartis), is a further example where health care system data from the UK's National Health System is being used to dramatically reduce trial costs by rapidly identifying patient populations through administrative data. This powerful collaboration among academic institutions, government and industry will accelerate development as well as potential market access through the National Institute for Health and Care Excellence's (NICE) approval programme and a population-level commercial arrangement to make it widely accessible to patients [START_REF] Iacobucci | Inclisiran: UK to roll out new cholesterol lowering drug from next year[END_REF] . International networks of clinical trials embedded into efficient health systems with such infrastructure would be a major asset.

Important efficiency gains may be derived from conducting trials directly with the patientclinican dyad rather than through dedicated trial sites or via specific investigators. This can be further enhanced by utilizing electronic data collection from wearables, and other data collection approaches such as point of care lab tests. Data quality is already dependent on remote monitoring and statistical fraud detection (trained AI algorithms) rather than solely by on-site monitoring visits with source data verification.

Working with key stakeholders towards global solutions

Despite advances in the processes and organisation of clinical guidelines and evidence-based medicine, which would be envisaged to promote the uptake and application of new therapies once proven in large scale Phase III randomised clinical trials, Pharma face a variety of diverse barriers unique to each nation in regard to federal regulatory authorities, and financial reimbursement policies. This requires a dedicated "army" of strategic company staff members who are often engaged in expensive lobbying with little logical relationship between the chance and rate of success and the clinical unmet need or demonstrated health and economic benefit. Whilst country-specific approaches will be required, moving beyond geographic borders will be essential. As healthcare globalizes, patients are increasingly demanding increased access to the latest innovations and are willing to go beyond their current clinical relationships to gain such access. The solutions likely to be effective in cardiovascular drug development are those seen in oncology where clinical trials are more tightly integrated into clinical care in a much more rapid cycle innovation framework. This will require transformation of the culture of cardiovascular care which often is balkanized on the basis of acuity and revenue for healthcare systems. Such culture change might best be focused on the creation of robust approaches to the implementation of existing proven therapies, which typically are deployed in a highly variable manner even a decade after reaching consensus endorsement by professional society clinical practice guidelines. Resolving these inequities alone would substantially reduce the cost of drug development, long before the advent of novel targets which would be pursued aggressively in tandem.

Community and government perceptions are often inaccurate, with many believing that much of cardiovascular disease is self-inflicted through poor lifestyle choices, or an inevitable part of ageing. This is compounded by the perception that the current armamentarium of drugs for atherosclerosis and heart failure already provide the entire solution. The remaining large gaps in our understanding and targeting of residual risk and missing mechanisms is ever important. A coordinated strategy engaging patients, clinicians, researchers, regulators and industry needs to be established to focus on strategies for the redesign of the cardiovascular drug discovery and development pipeline. Creating a community which provides a common voice to engage all stakeholders including governments, will be a critical first step and such an effort might rationally be convened by collaboration across the relevant professional and patient societies.

Next steps and concluding remarks

The COVID-19 Pandemic has provided the community with a poignant example of an acute health problem in urgent need of solutions. Understanding disease spread and individual susceptibility, as well as discovering new treatments and a vaccine are priorities immediately evident to all. The response of researchers from around the globe, working with health care, government leaders and industry, as well as the broader community, demonstrates the committed, diverse and innovative talent that can be mobilized to address our world's major health challenges. The extent of collaboration with industry, including between companies is also unprecedented [START_REF]CSL's Global Role in Battling COVID-19[END_REF] , yet the lack of any pre-existent structures within the biomedical ecosystem for global coordination against a pandemic some 100 years after the last example speaks to the core deficits at play when is not an immediate temporal imperative to manage a disease. It is important to remember that in the 6 months of COVID-19 to date, based on current statistics, ~9 million individuals around the globe will have died of cardiovascular disease, with the majority from low-and middle-income countries. The time is right to continue the momentum and international collaboration achieved to address COVID-19 and commit to taking similar steps and sustaining these in the fight against our greatest killer (Figure 2). Developing a global approach to transform the drug discovery and translation ecosystem for cardiovascular disease, whilst maintaining efforts towards equitable access to established effective treatments, is an imperative not an option.

Figure 2 .

 2 Figure 2. Call to action: collaborative next steps to accelerate discovery, translation and impact in cardiovascular medicine.

Table 1 .

 1 Challenges and potential solutions for accelerating drug discovery and translation in the cardiovascular space.

	Leadership and cultural shift to increased rigor,
	broader sharing of successes or failures and empiric
	evaluation of the translatability of the relevant
	models. Multicentre research networks for pre-
	clinical testing.

Disclosures

Dr. Figtree reports grants from National Health and Medical Research Council (Australia), personal fees from CSL, and grants from Abbott Diagnostic outside the submitted work. In addition, Dr. Figtree has a patent Biomarkers Dr. Zannad reports personal fees from Janssen, Novartis, Boston Scientific, Amgen, CVRx, other contributions from cardiorenal, personal fees from AstraZeneca, Vifor Fresenius, Cardior, Cereno pharmacuetical, Applied Therapeutics, Merck, other contributions from CVCT, personal fees from BAYER, Cellprothera, Owkin, Boehringer, Myokardia, Corvidia, and other contributions from G3 pharmaceuticals, outside the submitted work.


Christian A Devaux
email: christian.devaux@mediterranee-infection.com

Jean-Marc Rolain

Didier Raoult

ACE2 receptor polymorphism : susceptibility to SARS-CoV-2, hypertension, multi-organ failure, and COVID-19 disease outcome

Keywords: COVID-19, SARS-CoV-2, Hypertension, Cardiac failure, ACE2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged in Chinese people in December 2019 and has currently spread worldwide causing the COVID-19 pandemic with more than 150,000 deaths. In order for a SARS-CoV like virus circulating in wild life for a very long time to infect the index case-patient, a number of conditions must be met, foremost among which is the encounter with humans and the presence in homo sapiens of a cellular receptor allowing the virus to bind. Recently it was shown that the SARS-CoV-2 spike protein, binds to the human angiotensin I converting enzyme 2 (ACE2). This molecule is a peptidase expressed at the surface of lung epithelial cells and other tissues, that regulates the renin-angiotensin-aldosterone system. Humans are not equal with respect to the expression levels of the cellular ACE2. Moreover, ACE2 polymorphisms were recently described in human populations. Here we review the most recent evidence that ACE2 expression and/or polymorphism could influence both the susceptibility of people to SARS-CoV-2 infection and the outcome of the COVID-19 disease. Further exploration of the relationship between the virus, the peptidase function of ACE2 and the levels of angiotensin II in SARS-CoV-2 infected patients should help to better understand the pathophysiology of the disease and the multi-organ failures observed in severe COVID-19 cases, particularly heart failure.

Introduction

Over the past 20 years, seven coronaviruses responsible for more or less severe respiratory diseases have emerged in humans. Several of them, including SARS-CoV-2 (a Betacoronavirus lineage b/Sarbecovirus), can cause patients lung injury and sometimes multiorgan failure with adverse myocardial remodeling, myocardial stress, and cardiomyopathy [START_REF] Zhu | A Novel Coronavirus from Patients with Pneumonia in China, 2019[END_REF][START_REF] Huang | Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China[END_REF] .

Recently, SARS-CoV-2 was reported to be a human angiotensin I converting enzyme 2 (ACE2)-tropic virus [START_REF] Yan | Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2[END_REF][START_REF] Qiu | Predicting the angiotensin converting enzyme 2 (ACE2) utilizing capability as the receptor of SARS-CoV-2[END_REF] able to bind the alveolar pneumocytes which express ACE2 at their surface [START_REF] Zhao | Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov[END_REF][START_REF] Wang | Increasing host cellular receptor-angiotensinconverting enzyme 2 (ACE2) expression by coronavirus may facilitate 2019-nCoV infection[END_REF] . Yet, in humans the ACE2 mRNAs were found expressed in virtually all organs including the heart, blood vessels, kidney and testis, opening the possibility for this virus to infect other tissues beside lung [START_REF] Donoghue | A novel angiotensin-converting enzyme-related carboxypeptidase. (ACE2) converts angiotensin I to angiotensin 1-9[END_REF][START_REF] Tipnis | A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase[END_REF] . ACE2 is a known peptidase that regulates the reninangioten-aldosterone system (RAAS), thus controlling blood pressure. Therefore, it is not surprising that initials reports suggested that hypertension, diabetes and cardiovascular diseases were the most frequent comorbidity in COVID-19 disease [START_REF] Bavishi | Coronavirus Disease 2019 (COVID-19) Infection and Renin Angiotensin System Blockers[END_REF] .

The human coronaviruses

Coronaviruses (CoV) circulate in bats and generally pass over an intermediate animal host before crossing species barrier to infect humans [START_REF] Afelt | Bats, bat-borne viruses, and environmental changes[END_REF] . Different species of bats in China carry genetically diverse coronaviruses, some of which are direct ancestors of SARS-CoV [START_REF] Hu | Bat origin of human coronaviruses[END_REF][START_REF] Forni | Molecular Evolution of Human Coronavirus Genomes[END_REF][START_REF] Afelt | Bats, Coronaviruses, and Deforestation: Toward the Emergence of Novel Infectious Diseases?[END_REF] . Indeed, the first SARS-CoV that caused a human outbreak derived from SARS-like CoV circulating in Chinese horseshoe Rhinolophus bats which apparently adapted to wild Himalayan palm-civet before spreading in humans [START_REF] Song | Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human[END_REF] . The MERS-CoV originated from a Pipistrellus bat CoV and was probably transmitted to humans through contact with infected camels [START_REF] Wang | Bat origins of MERS-CoV supported by bat coronavirus HKU4 usage of human receptor CD26[END_REF][START_REF] Sabir | Co-circulation of three camel coronavirus species and recombination of MERS-CoVs in Saudi Arabia[END_REF][START_REF] Anthony | Further evidence for bats as the evolutionary source of Middle East respiratory syndrome coronavirus[END_REF] . Soon after the first outbreak of SARS-CoV-2 in humans, it was reported that this new virus was related to a bat-borne coronavirus (BatCoV RaTG13) present in the Rhinolophus affinis bat species [START_REF] Zhou | A pneumonia outbreak associated with a new coronavirus of probable bat origin[END_REF] . The identification of an intermediate animal hosts has been the subject of intense research and it was claimed that a pangolin (Manis javanica) was the intermediate host for SARS-CoV-2 [START_REF] Liu | Viral metagenomics revealed Sendai virus and coronavirus infection of Malayan pangolins (Manis javanica)[END_REF] . The SARS-CoV-2 receptor ACE2 from bat and pangolin and several other species, were found to resemble that of human [START_REF] Luan | Spike protein recognition of mammalian ACE2 predicts the host range and an optimized ACE2 for SARS-CoV-2 infection[END_REF] .

Before 2003, although human coronavirus 229E (HCoV-229E) (Alphacoronavirus) and

HCoV-OC43 (Betacoronavirus lineage a) described in the 1960s were known to be agents of respiratory infections, they lent little attention. In the early 2000s, two other coronaviruses responsible for similar diseases were identified, the HCoV-NL63 (Alphacoronavirus) and HCoV-HKU1 (Betacoronavirus lineage a). Even if the health authorities pay little attention to these viruses, sometimes they can cause deaths in people with fragile health. A study in Switzerland reported that among 279 subjects who had bronchoalveolar lavage for investigation of respiratory symptoms, 29 were tested positive for HCoV (detection rate: 10.4%) [START_REF] Garbino | A Prospective Hospital-Based Study of the Clinical Impact of Non-Severe Acute Respiratory Syndrome (Non-SARS)-Related Human Coronavirus Infection[END_REF] . A large-scale polymerase chain reaction (PCR) screening of 11,661 nasal samples from European patients with respiratory disease, found 35 HCoV-229E (0.30%), 61 HCoV-HKU1 (0.52%), 75 HCoV-NL63 (0.64%), and 111 HCoV-OC43 (0.85%) [START_REF] Gaunt | Epidemiology and clinical presentations of the four human coronaviruses 229E, HKU1, NL63, and OC43 detected over 3 years using a novel multiplex real-time PCR method[END_REF] . A similar study in Africa on 5,573 nasal samples from child hospitalized for pneumonia found 114 HCoV-229E (2.05%), 163 HCoV-NL63 (2.93%), and 111 HCoV-OC43 (1.99%) [START_REF] Kiyula | Human Coronavirus NL63 Molecular Epidemiology and Evolutionary Patterns in Rural Coastal Kenya[END_REF] . Two Chinese studies involving almost 25,000 throat and nasal swab samples from patients with acute respiratory tract infections revealed 114 HCoV-229E (0.37%-0.57%), 61 HCoV-HKU1 (0.18%-0.33%), 104 HCoV-NL63 (0.33%-0.52%), and 523 HCoV-OC43 (1.36%-3.04%), respectively [START_REF] Zeng | Epidemiology and clinical characteristics of human coronaviruses OC43, 229E, NL63, and HKU1: a study of hospitalized children with acute respiratory tract infection in Guangzhou, China[END_REF][START_REF] Zhang | Epidemiology characteristics of human coronaviruses in patients with respiratory infection symptoms and phylogenetic analysis of HCoV-OC43 during 2010-2015 in Guangzhou[END_REF] . The fatality rate of the coronaviruses causing the common winter cold was estimated 0.5% to 1.5% [START_REF] Choi | Comparison of the Clinical Characteristics and Mortality of Adults Infected with Human Coronaviruses 229E and OC43[END_REF] .

Coronaviruses strongly gained in notoriety when SARS-CoV (Betacoronavirus lineage b) emerged in China in March 2003 and was proven responsible for the severe acute respiratory syndrome (SARS) outbreak in humans [START_REF] Marra | The genome sequence of the SARS-associated coronavirus[END_REF] . The SARS-CoV adapted to humans and became able to spread from person-to-person leading to a fatality rate of 9.6% in infected patients, causing global concern. The Middle East Respiratory Syndrome (MERS) caused by the MERS-CoV (Betacoronavirus lineage 2c), was reported in Saudi Arabia in 2012. This epidemic which has been one of the least deadly in absolute number of deaths, was the one which has created the most fears in health authorities and the most important panic in the populations due to its high fatality rate (case fatality rate of 34.7%) [START_REF] Zaki | Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia[END_REF] . The SARS-CoV-2 that emerged in China at the end of 2019, is responsible for respiratory infections including pneumonia with a mortality rate estimated about 1%-2.5% 2 , increasing with age and the existence of underlying diseases. Under chest computerized tomography (CT) scans, the majority of patients show bilateral ground glass-like opacities and subsegmental areas of consolidation indicative of SARS-CoV-2 induced pneumonia.

The MERS-CoV, SARS-CoV, SARS-CoV-2 and their cellular receptors

Already for SARS-CoV, it was demonstrated that this virus used the angiotensin I converting enzyme 2 (ACE2) to enter human cells [START_REF] Ge | Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor[END_REF] . The novel Betacoronavirus SARS-CoV-2 (formerly 2019-nCoV), that cause COVID-19 disease, has 79.5% nucleotide identity with SARS-CoV 1 . It is worth noting that HCoV-NL63, SARS-CoV and SARS-CoV-2 spike proteins bind ACE2 [START_REF] Li | Angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis[END_REF] expressed at high levels in type I and II alveolar cells in the lung, whereas MERS-CoV bind the dipeptidyl peptidase 4 (DPP4)/CD26), a multifunctional serine peptidase known involved in T cell activation [START_REF] Wagner | Unravelling the immunological roles of dipeptidyl peptidase 4 (DPP4) activity and/or structure homologue (DASH) proteins[END_REF] . The analysis of SARS-CoV-2 spike (S) protein and ACE2 three-dimensional (3-D) structures allowed identification of regions in the peptidase domain of ACE2 required for viral spike binding [START_REF] Yan | Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2[END_REF] . Three very elegant papers published in the recent weeks characterized SARS-CoV-2 entry in target cells through interactions with ACE2 and serine protease TMPRSS2 priming as well as the 3-D structures involved in these interactions [START_REF] Yan | Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2[END_REF][START_REF] Hoffmann | SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor[END_REF][START_REF] Wrapp | Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation[END_REF] .

The human monocarboxypeptidase ACE-2 was originally cloned from human heart failure and lymphoma cDNA libraries [START_REF] Donoghue | A novel angiotensin-converting enzyme-related carboxypeptidase. (ACE2) converts angiotensin I to angiotensin 1-9[END_REF] . Although the ACE2 gene is usually considered silent in immune cells, the expression of ACE2 mRNAs was reported in a subset of CD14+ CD16human monocytes [START_REF] Rutkowska-Zapala | Human monocyte subsets exhibit divergent angiotensin I-convertingactivity[END_REF] . ACE2 is also expressed by enterocytes of the small intestine and expected to regulate the expression of the gut antimicrobial peptides [START_REF] Hashimoto | ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation[END_REF] . Moreover, this peptidase is also present on the arterial and venous endothelial cells, and arterial smooth muscle [START_REF] Hamming | Tissue distribution of ACE2 protein, the functional receptor for SARS Coronavirus[END_REF] . In normal human lung, the ACE2 protein is found on type I and II alveolar epithelial lung cells [START_REF] Sun | Understanding of COVID-19 based on current evidence[END_REF] . High expression of ACE2 was also reported on the epithelial cells of oral mucosa [START_REF] Xu | High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of orla mucosa[END_REF] . Single-cell RNA-seq analysis indicated that Asian men have a higher ACE2 mRNA expression in lung than women and that Asian people express higher amount of ACE2 than Caucasian and African American populations [START_REF] Zhao | Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov[END_REF] , but this observation remains controversial [START_REF] Cai | Tobacco-use disparity in gene expression of ACE2, the receptor of 2019-nCov[END_REF] . Until recently, the genetic basis of ACE2 expression in different populations remained largely unknown [START_REF] Cao | Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations[END_REF] .

ACE2 structure and function

The ACE2 gene span 39.98 kb of genomic DNA and contains 18 exons. It maps to chromosome X at position Xp22 8 . It encodes a type I cell-surface glycoprotein of about 100kDa, composed by 805 amino acids and characterized by a N-terminal signal peptide of 17 amino acid residues, a peptidase domain (PD) (residues 19-615) with its HEXXH zinc binding metalloprotease motif, a C-terminal Collectrin (a regulator of renal amino acid transport and insulin)-like domain (CLD) (residues 616-768) that includes a ferredoxin-like fold "Neck" domain (615-726), that end with an hydrophobic transmembrane hydrophobic helix region of 22 amino acid residues followed by an intracellular segment of 43 amino acid residues [START_REF] Donoghue | A novel angiotensin-converting enzyme-related carboxypeptidase. (ACE2) converts angiotensin I to angiotensin 1-9[END_REF][START_REF] Zhang | Collectrin, a Collecting Duct-specific Transmembrane Glycoprotein, Is a Novel Homolog of ACE2 and Is Developmentally Regulated in Embryonic Kidneys[END_REF] . The histidine motif HEXXH identified as an important component in a wide variety of zinc-dependent metalloproteases consists of five residues, the first histidine followed by glutamic acid being conserved, then the two variable amino acids and a final histidine [START_REF] Jongeneel | A unique signature identifies a family sequence, which could have better zinc-binding activity than of zinc-dependent metallopeptidases[END_REF] . Crystal structure analysis have suggested the presence of several hinge regions and N-glycosylations [START_REF] Watermeyer | Structure of Testis ACE Glycosylation Mutants and Evidence for Conserved Domain Movement[END_REF] .

ACE2 belongs to the family of ACE members which have a wider tissues distribution. The juxtamembrane, transmembrane and cytoplasmic tail of ACE2 do not resemble ACE but these two proteins share the CLD region, a 220 amino-acid domain. Angiotensin converting enzymes (ACE) are zinc metallopeptidases. ACE, is a widely distributed protein of 170 kDa encoded by a 21 kb gene located on chromosome 17 (17q23) [START_REF] Howard | Transcription of testicular angiotensin-converting enzyme (ACE) is initiated within the 12th intron of the somatic ACE gene[END_REF][START_REF] Sayed-Tabatabaei | ACE polymorphism[END_REF] , that converts the inactive decapeptide, angiotensin (Ang) I to an active vasoconstrictor octapeptide Ang II [Asp-Arg-Val-Tyr-Ile-His-Pro-Phe] that controls the blood pressure [START_REF] Erdos | The angiotensin I-converting enzyme[END_REF][START_REF] Ferrario | Counterregulatory actions of angiotensin-(1-7)[END_REF][START_REF] Oudit | The role of ACE2 in cardiovascular physiology[END_REF][START_REF] Turner | Exploring the structure and function of zinc metallopeptidases: old enzymes and new discoveries[END_REF] , and through inactivation of bradykinin vasodilatator [START_REF] Jaspard | Differences in the properties and enzymatic specifities of the two active sites of angiotensin I-converting enzyme (kininase II). Studies with bradykinin and other natural peptides[END_REF] . AngII also triggers the release of aldosterone that regulates the capacity of kidney to absorb sodium and water [START_REF] Brewster | The renin-angiotensin-aldosterone system and the kidney disease[END_REF] . Moreover, Ang II stimulates DPP4 activity likely via the seven-transmembrane receptor (7TM) angiotensin II type A receptor (AT1R)mediated transactivation of epidermal growth factor receptor [START_REF] Aroor | Angiotensin II Stimulation of DPP4 Activity Regulates Megalin in the Proximal Tubules[END_REF] and DPP4 inhibitors are described as a new class of anti-diabetic treatments the cardiovascular safety of which has been confirmed whereas their impact on hypertension is under evaluation [START_REF] Kawase | A dipeptidyl peptidase-4 inhibitor ameliorates hypertensive cardiac remodeling via angiotensin-II/sodiumproton pump exchanger-1 axis[END_REF] . Ang II also mediates cell proliferation by stimulating various cytokines [START_REF] Carluccio | Aspects of gene polymorphisms in cardiovascular disease : the renin-angiotensin system[END_REF] . ACE2, known for its diverse biological functions, including regulation of blood pressure through the renin-angiotensinaldosterone system (RAAS), converts the octapeptide AngII to the heptapeptide Ang(1-7) by hydrolysis of the C-terminal residue. Ang(1-7) is expect to exert its action through the MASrelated (MAS1) G protein-coupled receptor (GPGR) [START_REF] Santos | Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas[END_REF][START_REF] Karnik | Significance of angiotensin 1-7 coupling with MAS1 receptor and other GPGRs to the renin-angiotensin system: IUPHAR Review 22[END_REF] . In the pancreas ACE2 play an important glycemia-protective role [START_REF] Pedersen | The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs[END_REF] . Low ACE2 expression in the kidney is also associated with progressive renal diseases including diabetic nephropathy [START_REF] Reich | Decreased glomerular and tubular expression of ACE2 in patients with type 2 diabetes and kidney disease[END_REF] . A soluble form of the catalytic ACE2 ectodomain can be released in the circulation following cleavage between amino acids 716 and 741 by sheddase ADAM10 and ADAM17 [START_REF] Jia | Ectodomain Shedding of Angiotensin Converting Enzyme 2 in Human Airway Epithelia[END_REF][START_REF] Xiao | Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells[END_REF][START_REF] Devaux | The E-Cadherin Cleavage Associated to Pathogenic Bacteria Infections Can Favor Bacterial Invasion and Transmigration, Dysregulation of the Immune Response and Cancer Induction in Humans[END_REF] . The transcriptional regulation of ACE2 is under the control of DNA-binding protein such as Sirtuin 1 (SIRT1) [START_REF] Patel | Role of the ACE2/Angiotensin 1-7 axis of the Renin-Angiotensin System in Heart Failure[END_REF] .

(Figure 1A).

ACE2 limits the adverse vasoconstrictor and profibrotic effects of AngII. The hydrolysis of AngII into Ang (1-7) reduces the oxidative stress of AngII on endothelial cerebral arteries [START_REF] Silva | Impact of ACE2 deficiency and oxidative stress on cerebrovascular function with aging[END_REF] .

Ang(1-7) was reported to have vasodilatory and antifibrotic actions [START_REF] Tallant | Molecular mechanisms of inhibition of vascular growth by angiotensin-(1-7)[END_REF] . Disruption of ACE2 results in increased AngII levels and impaired cardiac function [START_REF] Crackower | Angiotensin-converting enzyme 2 is an essential regulator of heart function[END_REF] . Reduced levels of cardiac ACE2 have been reported in hypertension (HT) and diabetic heart disease [START_REF] Diez-Freire | ACE2 gene transfer attenuates hypertension-linked pathophysiological changes in the SHR[END_REF][START_REF] Tikellis | Interaction of diabetes and ACE2 in the pathogenesis of cardiovascular disease in experimental diabetes[END_REF] . Low expression of ACE2 mRNA was associated to HT, dyslipidemia and/or heart failure [START_REF] Velkoska | Angiotensin converting enzyme 2 and diminazene: role in cardiovascular and blood pressure regulation[END_REF] .

A polymorphism of ACE2 gene was first documented in the Chinese population with three ACE2 variants (rs4240157, rs4646155, and rs4830542) associated with HT [START_REF] Yi | Association of ACE, ACE2 and UTS2 polymorphisms with essential hypertension in Han and Dongxiang populations from North-Western China[END_REF][START_REF] Niu | Correlation of angiotensin-converting enzyme 2 gene polymorphisms with stage 2 hypertension in Han Chinese[END_REF][START_REF] Fan | Polymorphisms of angiotensin-converting enzyme (ACE) and ACE2 are not associated with orthostatic blood pressure dysregulation in hypertensive patients[END_REF][START_REF] Chen | Impact of ACE2 gene polymorphism on antihypertensive efficacy of ACE inhibitors[END_REF][START_REF] Luo | Association of ACE2 genetic polymorphisms with hypertension-related target organ damages in south Xinjiang[END_REF] , in a Nicotine Dependence in Teens Canadian cohort rs2074192, rs233575, and rs2158083 mutations were significantly associated with pathological variations of blood pressure [START_REF] Malard | The association between the angiotensin-converting enzyme-2 gene and blood pressure in a cohort study of adolescents[END_REF] . ACE2 rs21068809 mutation (C>T) has been reported associated with clinical manifestations of HT [START_REF] Chen | Correlation of angiotensin-converting enzyme 2 gene polymorphism with antihypertensive effects of benazepril[END_REF] . In Indian the study of 246 HT patients and 274 normotensive people indicated an association of HT with ACE2 rs21068809 mutation [START_REF] Patnaik | Association of angiotensin-converting enzyme and angiotensin-converting enzyme-2 gene polymorphisms with essential hypertension in the population of Odisha, India[END_REF] . In Brazilian patients, the combination of ACE I/D and ACE2 G8790A polymorphisms revels susceptibility to HT [START_REF] Pinheiro | The combination of ACE I/D and ACE2 G8790A polymorphisms revels susceptibility to hypertension: A genetic association study in Brazilian patients[END_REF] . The RAAS pathway can also be regulated by a polymorphism in ACE. In African-American with hypertension an ACE polymorphism was reported [START_REF] Duru | Frequency of a Deletion Polymorphism in the Gene for Angiotensin Converting Enzyme Is Increased in African-Americans With Hypertension[END_REF] .

Very recently, Cao and colleagues reported the results of a large investigation (1700 variants) of coding sequences variants in ACE2 and the allele frequency differences between populations in ACE2 gene from the China Metabolic Analytics Project and 1000 Genome Project database and other large scale genome databases [START_REF] Cao | Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations[END_REF] . They found one variant with a truncation Gln300 in China. In addition, they reported 32 variants among which seven hotspot variants in different populations.

Viral ACE2 receptor polymorphism and coronaviruses infection

It remains possible that ACE2 gene polymorphism, human ACE2 mRNA expression and human ACE2 protein polymorphism influence SARS-CoV-2 susceptibility and COVID-19 disease outcome.

For more than two decades, in the field of the human immunodeficiency virus (HIV), a retrovirus transmitted by sexual intercourse, it was demonstrated that the binding of the gp120 viral envelope glycoprotein to the CD4 receptor [START_REF] Benkirane | The cytoplasmic domain of CD4 plays a critical role during the early stages of HIV infection in T-cells[END_REF][START_REF] Briant | Binding of HIV-1 virions or gp120-anti-gp120 immune complexes to HIV-1-infected quiescent peripheral blood mononuclear cells reveals latent infection[END_REF] , to CXCR4 [START_REF] Biard-Piechaczyk | Caspase-dependent apoptosis of cells expressing the chemokine receptor CXCR4 is induced by cell membrane-associated human immunodeficiency virus type 1 envelope glycoprotein[END_REF][START_REF] Roland | Role of the intracytoplasmic domains of CXCR4 in SDF-1 mediated signaling[END_REF] or CCR5 coreceptor [START_REF] Alkhatib | HIV-1 Coreceptor Activity of CCR5 and Its Inhibition by Chemokines: Independence from G Protein Signaling and Importance of Coreceptor Downmodulation[END_REF] , triggers cell signaling. These molecules play a crucial role in the permanent molecular crosstalk between the cell and its environment. In this viral model, the study of the CCR5 coreceptor polymorphism clearly showed that a natural ∆32 deletion prevented the infection by HIV of homozygous people carrying this genotype [START_REF] Liu | Homozygous defect in HIV-1 corecptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection[END_REF][START_REF] Samson | Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene[END_REF] . For the MERS-CoV, attachment of the spike (S) glycoprotein to human cells require the host cell typeII transmembrane protein dipeptidyl peptidase 4 (DPP4/CD26) [START_REF] Raj | Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC[END_REF][START_REF] Wang | Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4[END_REF] . Following interaction with DPP4, the S protein of MERS-CoV undergoes proteolytic activation through the cellular serine protease TMPRSS2 and cysteine protease cathepsin L once inside endosomes [START_REF] Shirato | Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease TMPRSS2[END_REF] . Soluble forms of DPP4 can be released in the blood circulation after cleavage by the kallikrein-related peptidase 5 (KLK5) [START_REF] Nargis | KLK5 induces shedding of DPP4 from circulatory Th17 cells in type 2 diabetes[END_REF] . It was recently reported that among fourteen characterized mutants forms of DPP4, four polymorphisms (K267E, K267N, A291P and ∆346-348) strongly reduce the binding and penetration of MERS-CoV into target cells and the viral replication [START_REF] Kleine-Weber | Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus[END_REF] .

Regarding SARS-CoV, the S1 domain of the spike protein mediates ACE2 receptor binding whereas the S2 domain is a membrane-associated portion that likely undergoes post-binding transconformational modifications allowing membrane fusion. The viral receptor binding domain (RBD) located in S1 has been narrowed down to amino acid residues 318 to 510 [START_REF] Babcock | Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor[END_REF] . A co-crystal structure of ACE2 to the RBD revealed that residues 424 to 494 are involved in direct contact with the first α-helix and Lys353 and proximal residues at the N-terminus of βsheet 5 of ACE2 [START_REF] Li | Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2[END_REF] . By altering the His353 amino acid in rat ACE2 and modifying a glycosylation site (Asp 90) that may alter the conformation of the α-helix 1 of ACE2, Li and colleagues [START_REF] Li | Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2[END_REF] converted the rat ACE2 into an efficient receptor for SARS-CoV. A point mutation Leu584Ala in ACE2, markedly attenuated the shedding of the enzyme and facilitated SARS-CoV entry into target cells [START_REF] Xiao | Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells[END_REF] . A soluble form of ACE2 lacking the cytoplasmic and transmembrane domain of the molecule was reported capable of blocking binding of SARS-CoV spike protein to ACE2 [START_REF] Lambert | Tumor Necrosis Factor-Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2)[END_REF] . Expression of ACE2 was found down regulated in cells infected by SARS-CoV [START_REF] Kuba | A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury[END_REF] . A recombinant SARS-CoV spike protein was found to down regulated ACE2 expression through release of sACE2 and thereby promotes lung injury [START_REF] Glowacka | Differential Downregulation of ACE2 by the Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus and Human Coronavirus NL63[END_REF] . Among other antiviral effect of Chloroquine on SARS-CoV in vitro one could be attributable to a deficit in the glycosylation of the ACE2 virus cell surface receptor [START_REF] Vincent | Chloroquine is a potent inhibitor of SARS coronavirus infection and spread[END_REF][START_REF] Devaux | New insights on the antiviral effects of chloroquine against coronavirus: what to expect for COVID-19?[END_REF] .

Regarding the HCoV-NL63 that also employ ACE2 for cell entry a recombinant SARS-CoV/HCoV-NL63 spike protein trigger shedding of sACE2 [START_REF] Burrell | The ACE2 Gene: Its Potential as a Functional Candidate for Cardiovascular Disease[END_REF] .

Very recently, investigation of SARS-CoV-2 cell entry through ACE2 binding showed important commonalities between SARS-CoV and SARS-CoV-2 infection, including similar choice of entry receptors [START_REF] Hoffmann | SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor[END_REF] . SARS-CoV and SARS-CoV-2 share about 76% amino acids identity and most amino acid residues essential for ACE2 binding were conserved in the SARS-CoV-2 spike S1 domain. Another recent paper published reported the structural basis of SARS-CoV2 interaction with ACE2 3 . The trimeric SARS-CoV-2 S1 spike binds the PD domain of ACE2 and the cleavage of ACE2 C-terminal segment (residues 697 to 716) by the transmembrane protease serine 2 (TMPRSS2) enhances the S-protein-driven viral entry. By comparing the 805 amino acid residues of the 10 human ACE2 proteins and the 4 different ACE2 isoforms available through GeneBank using Clustal Omega multiple sequence alignment, a 100% identity among the complete ACE2 sequences was observed and the isoforms corresponded to a deletion in the CLD domain, or truncation in the transmembrane domain. The role of these isoforms in SARS-CoV-2 infection and COVID-19 outcome, remains speculative. According to the recent work by Cao and colleagues [START_REF] Cao | Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations[END_REF] , 32 variants of ACE2 where characterized among which seven hotspot variants (Lys26Arg, Ile486Val, Ala627Val, Asn638Ser, Ser692Pro, Asn720Asp, and Leu731Ile/Phe) in different populations (Figure 1B). This open the possibility that some people could be less susceptible to SARS-CoV-2 infection than others.

Discussion

ACE2 protein at the surface of lung alveolar epithelial cells allows infection of the respiratory tract with SARS-CoV-2. It can be hypothesized that the ACE2 levels correlate with susceptibility to SARS-CoV-2 infection. Apparently, men have a higher ACE2 expression in lung than women and Asian people express ACE2 higher than Caucasian and African American populations [START_REF] Sun | Understanding of COVID-19 based on current evidence[END_REF] . This is in agreement with the finding that conversion of Ang II to Ang (1-7) by ACE2 was higher in males than female 100 , suggesting an over-expression of ACE2 in men. Because ACE2 is encoded by a gene located on the X chromosome and men express more ACE2 than women it could be speculated that depending the allele expressed by women, they could be considered of lower sensitivity against the most severe adverse effects of the infection [START_REF] Burrell | The ACE2 Gene: Its Potential as a Functional Candidate for Cardiovascular Disease[END_REF]101 . All clinical reports published to date indicate that men represent between 66% and 75% of the most severe cases of COVID-19. During early SARS-CoV-2 infection and viral spread within body tissues, the ACE2 function is likely impaired either by steric hindrance of the peptidase domain of ACE2 following virus binding or by down regulation of ACE2 mRNA expression and ACE2 protein. In severe COVID-19 disease, the presence of the viral receptor on other tissues than lung may explain the multi-organ failure sometimes observed in clinic. We therefore suggest that quantification of ACE2 and AngII be added to the COVID-19 patients biological monitoring.

It is known that ACE2 can shift the RAAS balance by conversion of Ang II to Ang (1-7).

Consequently, HT and COVID-19 recently become a question of concern for international professional societies of cardiology regarding: i) the susceptibility of patients with HT to get COVID-19; ii) the severity of the disease; and, iii) the use of ACE inhibitors (ACEi) and AngII receptor blockers (ARBs, that targets the AT1R). It is known that HT inhibitors increase the cell-surface expression of ACE2. It was demonstrated that ACEi can increase intestinal ACE2 mRNA expression 102 . Although data are lacking regarding the effects of such drugs on ACE2 mRNA expression in lung epithelial cells, there is a concern that patients taking those treatments can favor virus capture. In patients with HT who received long-term olmesartan (ARB) treatment, urinary ACE2 levels were higher than among untreated control patients 103 . In contrast to HT, in patients suffering from idiopathic pulmonary fibrosis, the expression levels of ACE2 are markedly decreased [START_REF] Li | Angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis[END_REF] . ACE2 is a major actor toward resolution of inflammation and fibrosis 104 . In an animal model of bleomycin-induced pulmonary fibrosis, treatment with intraperitoneal injection of recombinant human ACE2 improved the lung function and decreased lung inflammation and fibrosis 105 . Moreover, impaired phosphorylation of ACE2 Ser680 by AMP-activated protein kinase in pulmonary endothelium leads to a labile ACE2 and hence pulmonary HT 106 . We must also paid attention to molecules such as xanthenone (XNT) and dimiazene aceturate (DIZE, an antitrypanosomal drug) described as ACE2 activators 107 . In a rat model of ischemic heart disease, the subcutaneous infusion of DIZE significantly increased cardiac ACE2 mRNA expression and ACE2 protein catalytic activity, reduced ACE mRNA expression, and improved cardiac remodeling 108 . The possible beneficial properties of other molecules such as exenatide (a glucagon-like peptide-1 agonist) which induces an increase in vasodilatory and a decrease in vasoconstrictive mediators must also be investigated 109 . In addition, it was recently reported that heparin (anticoagulant) treatment is associated with decreased mortality in severe COVID-19 patients with coagulopathy 110 .

In a Chinese cohort of 1,099 patients with COVID-19, 165 (13%) individuals were patients with HT, among which 24% suffered from severe COVID-19, a percentage of 3.7%, slightly higher to that of the general population of COVID-19 patients 111 . In a smaller cohort of 191 patients with COVID-19, 58 (30%) were patients with hypertension and 48% of them died, which is surprisingly high percentage (14.6%) 112 . These results suggest that the prevalence of patients with HT was higher in patients who developed severe COVID-19 disease than those who do not. By mid march 2020, the international professional societies of cardiology recommended continuing patients' treatment 113 . Indeed, when the SARS-CoV-2 spike binds its ACE2 receptor in the α helix 1 (Lys31, Tyr 41) and β5 region (Lys353) it likely reduces the catalytic properties of ACE2 that is usually associated with reduced inflammation. The lack of Ang(1-7) generation may increase lung injury 114,115 and cardiovascular risks, Ang II acting like an inflammatory cytokine 116 . In a murine model it was observed that lung inflammation aggravates AngII-induced induced abdominal aortic aneurysms 117 .

Mutations might modify the expression level of ACE2 protein as shown in a murine model 118 . The deletion of ACE2 in mice model was associated with increased circulation and tissue AngII levels and led to cardiovascular damage 119,120 . It remain possible that i) mutations affecting the human ACE2 gene; ii) transcriptional variation in ACE2 mRNA expression; iii) post-transcriptional modifications that act on the ACE2 viral receptor (such as Nglycosylation), and; iv) putative ACE2 protein mutations, may influence the outcome of COVID-19 by acting on blood pressure through the RAAS and possible increasing of lung and heart damages through the oxidative stress triggered by Ang II. Recently an high rate fatality of SARS-CoV-2 was reported in Iran 121 , without satisfactory explanation. If underreporting of the number of infected people can be excluded, it could be hypothesized: i) a more aggressive variant clade of SARS-CoV-2 122 ; ii) a variation in ACE2; or, iii) a variation in genes like those encoding Toll-like receptors. Since it is known that Ang (1-7) prevents inflammation by inhibiting the resistin/ Toll-like receptor 4 (TLR4)/MAPK/NF-kB pathway 123 and that there is a high variability of the TLR4 gene in different ethnic groups in Iran 124 , it remains possible that SARS-CoV-2 triggers increased inflammation in Iranian patients by suppressing the ACE2-mediated metabolism of AngII to Ang (1-7). This could be related to the observation that mice deficient in the TLR3/TLR4 adaptor TRIF are highly susceptible to SARS-CoV infection including severe inflammatory induction 125 .

The mechanism of acute myocardial injury caused by SARS-CoV-2 during severe COVD-19 disease might be related to the inhibition of ACE2 catalytic activity 126 . (Figure 2).

Interestingly, a recent study posted as a pre-print paper 127 pointed out a list of 97 approved drugs that may have a therapeutic potential against COVID-19 including anti-diabetics (metaformin), statins (simvastatin) and ARBs (sartans). Medical records of patients currently treated with these compounds may help to identify whether those drugs have a beneficial or adverse effect on COVID-19 patients. Metaformin was also identified as a potential drugrepurposing against SARS-CoV-2 in another study 128 . It should be remembered that a large number of data suggest that there is a mild or severe cytokine storm in severe COVID-19 patients which is an important cause of death. To reduce the pro-inflammatory effect of AngII and the cytokine storm observed in severe cases of COVID-19, it might make sense to continue treating patients with ACE inhibitors and ARBs, a conclusion shared by recent recommendations of the international societies of cardiology 129 . However, Fang and colleagues 115 recently reported that the most distinctive comorbidities in patients who died from COVID-19 are HT, coronary heart diseases, cerebrovascular diseases and diabetes, and among them several were treated by ACE inhibitors. How should clinicians navigate this uncertainty for patients who are taking ACE inhibitors and ARBs and become infected with SARS-CoV-2? Do these molecules have a harmful effect in the outcome of the disease or is the link that is made highlights only a confounding factor which confirms that HT is a major factor of comorbidity? In agreement with others 130-132 , we consider that it is of special importance to rapidly evaluate whether these drugs are more beneficial than harmful in severe COVID-19 patients.

Figure 1 1A.

 1 Figure 1 1A. Schematic representation of the regulation of ACE2. The transcription of the Ace2 gene is under control of the SIRT1 DNA-binding protein that binds the Ace2 gene promotor. Posttranscriptional regulation by miRNA (miRNA143, miRNA421) could occur (not shown). Following translation the newly synthesized ACE2 proteins are likely target of posttranscriptional modifications such as phosporylation of Ser680 by AMPK that enhances the stability of ACE2, and N-glycosylations. Once expressed at the cell membrane the ACE2 protein can be regulated by sheddases (ADAM10, ADAM17) that cleave the ACE2 extracellular domain and release a circulating soluble form sACE2 capable to interact with integrins (ITGB1). 1B. Schematic representation (left) of the ACE2 molecule and its major domains. The amino acids position is in black. Some of the amino acids important for viral tropism are in red (previous studies showed that residues 31, 41, and regions 82-84 and 353-357 are important for viral spike binding). Clustal Omega multiple sequence alignment (EMBL-EBI bioinformatic tool; Copyright © EMBL 2020) of human ACE2 and its different isoforms (right). The comparison of the reference Homo sapiens ACE2 protein sequence (S1=Genbank: BAB40370.1) with 9 others ACE2 sequences from the NCBI reference sequence database (S2=UniProtKB Q9BYF1.2; S3=NCBI NP_001358344.1; S4=NCBI NP_068576.1; S5= GenBank EAW98892.1; S6= GenBank AAH48094.2; S7= GenBank AAH39902.1; S8= GenBank AAO25651.1; S9= GenBank BAD99267.1; GenBank AAF99721.1), showed 100% amino acids identity (not shown). The Clustal MSA was also used for the comparison of the human ACE2 S1 sequence and available sequences of ACE2 isoforms: the isoform X1 (I1)= NCBI XP_011543851.1; isoform X2 (I2)= NCBI XP_011543853.1; isoform X3(I3)=NCBI XP_011543854.1; isoform CRA (I4)=GenBank EAW98891.1. The figure illustrates that these isoforms correspond to deletions in the CLD domain, or truncations in the transmembrane domain. A very elegant work by Cao and colleagues 42 has recently analyzed 1700 ACE2 variants in search of ACE2 protein polymorphism. The mutations and truncations found by this team are shown in light blue.

Figure 2

 2 Figure 2 Simplified diagram of the renin-angiotensin system in normal and pathologic conditions. The left panel indicates that ACE2 converts Ang II to Ang(1-7) leading to protective signal. The right panel illustrates the possible dysfunction of signals when SARS-CoV-2 is attached to its ACE2 receptor. Under this condition Ang(1-7) is no longer synthetized, Ang II accumulates and binds AT1R, leading to proinflammatory signals that trigger both tissues damage (in particular lung and heart) and hypertension.

Funding

This work was supported by the French Government under the « Investissements d'avenir » (Investments for the Future) program managed by the Agence Nationale de la Recherche (French ANR: National Agency for Research), (reference: Méditerranée Infection 10-IAHU-03).

Competing Interests

CD declare a link of interest with the Sanofi and Merck pharmaceutical companies. JMR and DR declare that they have no competing interests.

Ethical Approval

Not required