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DECLARATION OF CANADIAN PHYSICIANS FOR SCIENCE AND TRUTH

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Source: https://canadianphysicians.org/

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The Declaration

We are a broad and diverse group of Canadian physicians from across Canada who are sending out this urgent declaration to the Colleges of Physicians and Surgeons of our various Provinces and Territories and to the Public at large, whom we serve.

On April 30, 2021, Ontario’s physician licensing body, the College of Physicians and Surgeons of Ontario (CPSO), issued a statement forbidding physicians from questioning or debating any or all of the official measures imposed in response to COVID-19. 1

The CPSO then went on to threaten physicians with punishment – investigations and disciplinary action.

We regard this recent statement of the CPSO to be unethical, anti-science and deeply disturbing.

As physicians, our primary duty of care is not to the CPSO or any other authority, but to our patients. 

When we became physicians, we pledged to put our patients first and that our ethical and professional duty is always first toward our patients. The CPSO statement orders us to violate our duty and pledge to our patients in the following ways:

1. Denial of the Scientific Method itself:  The CPSO is ordering physicians to put aside the scientific method and to not debate the processes and conclusions of science.

We physicians know and continue to believe that throughout history, opposing views, vigorous debate and openness to new ideas have been the bedrock of scientific progress.  Any major advance in science has been arrived at by practitioners vigorously questioning “official” narratives and following a different path in the pursuit of truth.

2. Violation of our Pledge to use Evidence-Based Medicine for our patients:  By ordering us not to debate and not to question, the CPSO is also asking us to violate our pledge to our patients that we will always seek the best, evidence-based scientific methods for them and advocate vigorously on their behalf.

The CPSO statement orders physicians for example, not to discuss or communicate with the public about “lockdown” measures. Lockdown measures are the subject of lively debate by world-renown and widely respected experts and there are widely divergent views on this subject. The explicitly anti-lockdown Great Barrington Declaration – https://gbdeclaration.org – was written by experts from Harvard, Stanford and Oxford Universities and more than 40,000 physicians from all over the world have signed this declaration. Several international experts including Martin Kuldorf (Harvard), David Katz (Yale), Jay Bhattacharya (Stanford) and Sunetra Gupta (Oxford) continue to strongly oppose lockdowns.

The CPSO is ordering physicians to express only pro-lockdown views, or else face investigation and discipline. This tyrannical, anti-science CPSO directive is regarded by thousands of Canadian physicians and scientists as unsupported by science and as violating the first duty of care to our patients.

3. Violation of Duty of Informed Consent:  The CPSO is also ordering physicians to violate the sacred duty of informed consent – which is the process by which the patient/public is fully informed of the risks, benefits and any alternatives to the treatment or intervention, before consent is given.

The Nuremberg Code, drafted in the aftermath of the atrocities perpetrated within the Nazi concentration camps – where horrific medical experiments were performed on inmates without consent – expressly forbids the imposition of any kind of intervention without informed consent.

In the case of the lockdown intervention for example, physicians have a fiduciary duty to point out to the public that lockdowns impose their own costs on society, including in greatly increased depression and suicide rates, delayed investigation and treatment of cancer (including delayed surgery, chemotherapy and radiation therapy), ballooning surgical waiting lists (with attendant greatly increased patient suffering) and increased rates of child and domestic abuse.

We physicians believe that with the CPSO statement of 30 April 2021, a watershed moment in the assault on free speech and scientific inquiry has been reached.

By ordering physicians to be silent and follow only one narrative, or else face discipline and censure, the CPSO is asking us to violate our conscience, our professional ethics, the Nuremberg code and the scientific pursuit of truth.

We will never comply and will always put our patients first.

The CPSO must immediately withdraw and rescind its statement of 30 April 2021.

We also give notice to other Canadian and international licensing authorities for physicians and allied professions that the stifling of scientific inquiry and any order to violate our conscience and professional pledge to our patients, itself may constitute a crime against humanity.

1 College of Physicians and Surgeons of Ontario Statement on Public Health Misinformation (4/30/21). https://twitter.com/cpso_ca/status/1388211577770348544

The College is aware and concerned about the increase of misinformation circulating on social media and other platforms regarding physicians who are publicly contradicting public health orders and recommendations. Physicians hold a unique position of trust with the public and have a professional responsibility to not communicate anti-vaccine, anti-masking, anti-distancing and anti-lockdown statements and/or promoting unsupported, unproven treatments for COVID-19. Physicians must not make comments or provide advice that encourages the public to act contrary to public health orders and recommendations. Physicians who put the public at risk may face an investigation by the CPSO and disciplinary action, when warranted. When offering opinions, physicians must be guided by the law, regulatory standards, and the code of ethics and professional conduct. The information shared must not be misleading or deceptive and must be supported by available evidence and science.

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Rebuttal letter to European Medicines Agency from Doctors for Covid Ethics, April 1, 2021

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Source: Doctors for Covid Ethics

Doctors, scientists, lawyers and colleagues in allied disciplines can sign the open letter by sending their name, qualifications, areas of expertise and country of practice to: Doctors4CovidEthics@protonmail.com, with web verification (eg workplace or registration link, not for publication).

Apr 1·9 min read

Emer Cooke, Executive Director, European Medicines Agency, Amsterdam, The Netherlands

April 1st, 2021

Ladies and Gentlemen,

FOR THE URGENT PERSONAL ATTENTION OF: EMER COOKE, EXECUTIVE DIRECTOR OF THE EUROPEAN MEDICINES AGENCY

We acknowledge receipt of your March 23 reply to our letter dated February 28, seeking reassurance that foreseeable risks of gene-based COVID-19 “vaccines” had been ruled out in animal trials prior to human use. Our concerns arise from multiple lines of evidence, including that the SARS-CoV-2 “spike protein” is not a passive docking protein, but its production is likely to initiate blood coagulation via multiple mechanisms.

Regrettably, your reply of March 23 is unconvincing and unacceptable. We are dismayed that you choose to respond to our request for crucially important information in a dismissive and unscientific manner. Such a cavalier approach to vaccine safety creates the unwelcome impression that the EMA is serving the interests of the very pharmaceutical companies whose products it is your pledged duty to evaluate. The evidence is clear that there are some serious adverse event risks & that a number of people, not at risk from SARS-CoV-2, have died following vaccination.

1. You concede that the “vaccines”, which are more accurately described as investigational gene-based agents, enter the bloodstream but you can obviously provide no quantitative data. In the absence of the latter, any scientific assessment you purport to have undertaken lacks foundation.

2. Your statement that non-clinical studies do not indicate any detectable uptake of the vaccines into endothelial cells lacks credibility. We demand to see the scientific evidence. If not available, it must be assumed that endothelial cells are targeted.

3. Auto-attack could not have been excluded in animals unless they had been immunologically primed beforehand. We demand evidence that such experiments had been performed. Similar experiments have been undertaken before with previous, unsuccessful candidate vaccines, and fatal, antibody-dependent enhancement of disease was observed.

4. We requested scientific evidence, not a vague description of what was purportedly seen in non-valid animal experiments. Your cursory mention of laboratory findings in humans is cynical. In view of the plausible connection between production of spike protein and the emergence of thromboembolic serious adverse events (SAEs), we demand to see the results of D-dimer determinations. As you are aware, D-dimer is a very good test as an aid to diagnose thrombosis.

After delivery of our letter to you on March 1, events followed that debunk your response to our last three queries to an extent that can only be termed embarrassing. As we feared, severe and fatal coagulopathies occurred in young individuals following “vaccination”, leading 15 countries to suspend their AZ-“vaccination” program. An official investigation by the EMA into the cases of afflicted younger individuals followed, the results of which were announced by the WHO on March 17, 2021, stating: “At this time, WHO considers that the benefits of the AstraZeneca vaccine outweigh its risks and recommends that vaccinations continue.”

What was this decision based upon? The WHO is not a competent body for formally evaluating drug safety. That is explicitly the role of the agency you lead.

In your press release, you disclosed the following information to support your conclusion. You had scrutinized data on two mortally dangerous conditions that had followed within 14 days of “vaccination”: DIC, disseminated intravascular coagulation; and CSVT, cerebral sinus vein thrombosis. 5 DIC and 18 CSVT were on record, with a total death toll of 9. Most cases were <55 year-old individuals. 5 DIC and 12 CSVT were under 50 years of age. None were reported as having had serious pre-existing illness.

You stated numbers that “normally” would be expected : DIC <1, CSVT 1.3.

Consequently, for these very rare conditions, a link to vaccination could not entirely be dismissed. However, given that 20 million individuals had been “vaccinated”, the benefits were deemed to far outweigh the risks.

But in fact, your Press Release rendered it glaringly apparent that the AZ-“vaccine” does have the potential to trigger intravascular coagulation, that the true risks far outweigh any theoretical benefits, and that any authority with the slightest sense of responsibility must suspend its further use.

1. Regard your incidence numbers for <50 year old individuals in the “vaccinated” versus “normal” population:

CSVT : 12 versus 1.3.

A 9-fold increase is beyond the range of coincidence.

DIC : 5 versus <1.

As we hope you know, DIC neveroccurs out of the blue in healthy individuals. The incidence should not be stated as <1 when in reality it is ZERO.

ACCORDINGLY, THE DIC CASES REPRESENT CONCLUSIVEEVIDENCE THAT THE AZ-VACCINE ALONE CAN TRIGGER INTRAVASCULAR COAGULATION .

2. Assume that 10 million recipients of the “vaccine” were < 60 yrs and this was followed by 9 deaths due to DIC and SVCT. The death toll upon 60 million “vaccinations” would be extrapolatable to 54.

The pandemic hit around 60 million individuals < 60 yrs in Germany.

During the first 6 months it reportedly claimed 52 lives of individuals without pre-existing illness (https://www.rki.de/DE/Content/Gesundheitsmonitoring/JoHM/2020/JoHM_Inhalt_20_S11.html)

Because of the unreliability of PCR testing and because of the completely novel way that deaths ‘with covid19’ are determined, the value of 52 is an over-estimate of the real burden of disease, further weakening your already-inadequate claim for risk-benefit.

How, then, can you declare that the benefits of vaccination far outweigh the risks? We demand your reply supported by facts and figures that we will convey to the public.

3. Further considerations expose the truly frightful dimensions of your irresponsible assertion.

CSVT, cerebral venous thrombosis, is always a life-threatening condition that demands immediate medical attention. The number of cases you conceded had occurred can represent just the tip of a huge iceberg. As you must know, the most common symptoms of CSVT are piercing headache, blurred vision, nausea and vomiting. In severe cases, stroke-like symptoms occur including impairment of speech, vision and hearing, body numbness, weakness , decreased alertness and loss of motoric control.

Surely, you are not oblivious to the fact that countless individuals suffered from precisely such symptoms directly following “vaccinations” with all the experimental gene-based agents.

Clot formation in deep leg veins can lead to lethal pulmonary embolisms. Surely you must know that peripheral venous thromboses have repeatedly been reported following “vaccinations” with all the experimental gene-based agents

Microthromboses in the lung vasculature can lead to misdiagnosis of pneumonia. In combination with false-positive PCR (with high cycle thresholds), these will then be registered as COVID 19 cases. Surely you must know that this scenario has probably repeatedly taken place following “vaccinations” with all the experimental gene- based agents.

In all events, extensive thrombi formation can lead to consumption of platelets and coagulation factors, resulting in hemorrhagic diathesis and bleeding at all possible locations. Surely you must know that profuse skin bleedings have repeatedly been observed following “vaccinations” with all the experimental gene-based agents.

Given that there is a mechanistically plausible explanation for these thromboembolic adverse drug reactions (TE ADRs), namely that the gene-based products induce human cells to manufacture potentially pro-thrombotic spike protein, the reasoned & responsible assumption must now be that this may be a class effect. In other words, the dangers must be ruled out for all emergency-authorised gene-based vaccines, not merely the AZ product.

We urge you to adopt this stance unless and until there is data providing high clinical confidence to the contrary. We are very willing to liaise with the Agency in order to help craft a focussed pharmacovigilance plan to accomplish this goal. With the above in mind, we hope you are aware that all thrombotic events can be rapidly diagnosed by measurement of D-Dimers in blood. And that good medical practice imperatively demands that attempts are undertaken to diagnose CSVT in any and every patient, young or old, presenting with the typical signs and symptoms following “vaccination”.

Given the potential for adverse effects, potentially fatal ones, it is completely inappropriate and unacceptable that EMA permits these products, which hold only emergency use authorisations, to be administered to younger (<60y) people who are healthy, as they are at unmeasurable risks from SARS-CoV-2.

Not to make this explicit is, in our view, a reckless stance to have taken in the first place and doubly so now.

Of equal importance, you are bound by duty to investigate whether reasons exist for the waves of deaths that have occurred following “vaccination” of elderly residents in care and senior homes. Or are you asserting that dangers of “vaccine”-derived thrombotic events are limited to younger individuals? If not, restricting their use solely in one age group — as decided upon in Germany — equates with nothing less than monstrous, condoned genocide of the other.

In closing, failure to inform “vaccine” recipients of the risks and negligible benefits outlined here represents serious violations of medical ethics and citizens’ medical rights. Those violations are especially grave as all the risks we describe can be expected to increase with each re-vaccination, and each intervening coronavirus exposure. This renders both repeated vaccination and common coronaviruses dangerous to young and healthy age groups, for whom — in the absence of “vaccination” — COVID-19 poses no substantive risk.

Such is the real risk-benefit analysis of the COVID-19 “vaccines”. Either the EMA lacks the subject-matter expertise to appreciate the molecular science of this reality, or it lacks the medical ethics to act accordingly.

At best, we regard the EMA’s complacent stance on vaccine dangers to be symptomatic of the fact that, under the prevailing politico-medical response to COVID-19, medical ethics has migrated from the consulting room to a geopolitical stage. Faced with a medical problem, mass-medical intervention has seen the practice of medicine taken from doctors’ hands. In this politicized context, corporate and political actors may consider themselves free from ethical constraints, operating unbound by a medical code of ethics, unlike medical doctors. All actors, however, are bound by the Nuremberg Code.

The Nuremberg Code prohibits human experimentation of the very kind being endorsed and defended by the EMA. Even under the terms of their own original FDA authorization, COVID-19 vaccines are deemed “investigational” and their recipients “human subjects”, who are, by definition, entitled to informed consent. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/emergency-use-investigational-drug-or-biologic#:~:text=Emergency%20use%20is%20defined%20as,21%20CFR%2056.102(d)%5D.

Misleading populations into accepting investigational agents such as the gene-based COVID-19 “vaccines”, or coercing them through “vaccine passports”, constitutes clear and egregious violations of the Nuremberg Code. The Nuremberg Code mandates voluntary informed consent “without the intervention of any element of force, fraud, deceit [or] duress”. https://history.nih.gov/display/history/Nuremberg+Code

In other words, citizens have the right under the Nuremberg Code and related protections not to be subject involuntarily to medical experiments. It is clear that these experimental agents should be CONTRA-INDICATED in individuals not at elevated risk of serious illness & death if infected by SARS-CoV-2. Furthermore, the use of the experimental agents must also be withheld in the elderly population until a risk-benefit assessment has been properly conducted. In any event, the vaccine label must be revised to reflect the recently emerged serious adverse events addressed here.

We remind the EMA that Nuremberg violations constitute crimes against humanity under the Geneva Convention. Crimes against humanity are deemed “the worst atrocities known to mankind”, and are prosecuted under the Rome Statute of the International Criminal Court. https://www.un.org/en/chronicle/article/role-international-criminal-court-ending-impunity-and-establishing-rule-law

Given the hundreds of millions and eventually billions of people who may be coerced into accepting these agents, the EMA, in persistently shrinking from open debate and the truth, will be seen by lawyers and historians as having actively assisted in crimes against humanity, with the full weight of the implications to all involved. We demand thatyou engage openly with us to ensure that the public have an objective understanding of the clinical risk profile of these gene-based interventions.

You understand that coercive pressure is being placed on citizens to receive COVID-19 vaccines, which are experimental medical treatments. Your responsibility to those citizens includes ensuring that they are informed of the adverse event risks of every such treatment. To date you have failed to do so, and have instead misled the public on the reality of the “vaccines’” risk-benefit profile.

If you continue to conceal the truth, efforts will be made to bring this to light and to see that justice is done. For the sake of the injured and the dead, and to protect further lives from similar fates.

NOTICE

For the avoidance of doubt, if your regulatory body does not immediately suspend its “emergency” recommendation of potentially dangerous inadequately tested gene-based “vaccines”, while the matters which we have highlighted to you are properly investigated, we hereby put the European Medicines Agency on notice of being complicit in medical experimentation, in violation of the Nuremberg Code, which thereby constitutes the commission of crimes against humanity.

Furthermore, it is your indirigible duty as a regulatory body to ensure that all doctors worldwide are advised that they are taking part in medical experimentation via “vaccination” programmes, whether wittingly or unwittingly, with all the legal and ethical obligations that such involvement entails.

This email is copied to the lawyer Reiner Fuellmich. It is also copied to Charles Michel, President of the Council of Europe, and to Ursula von der Leyen, President of the European Commission.

Yours faithfully,

Doctors for Covid Ethics

Doctors, scientists, lawyers and colleagues in allied disciplines can sign the open letters by sending their name, qualifications, areas of expertise and country of practice to: Doctors4CovidEthics@protonmail.com, with web verification (eg workplace or registration link, not for publication).

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Perspectives on the Pandemic | “Blood Clots and Beyond” | Episode 15 | Journeyman Pictures

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Norm’s note: in addition, see this debriefing (in both a dubbed French version and an original English version) with Professor Sucharit Bhakdi, M.D.

From the YouTube summary:

In February, 2021, Professor Sucharit Bhakdi, M.D. and a number of his colleagues warned the European Medicines Agency about the potential danger of blood clots and cerebral vein thrombosis in millions of people receiving experimental gene-based injections.

Since then, two of the four injections have been suspended or recalled in Europe and the United States for just that reason.

In this episode of Perspectives, Professor Bhakdi explains the science behind the problem, why it is not just limited to the products already suspended, and why in the long term we may be creating dangerously overactive immune systems in billions of unwitting subjects.

Subscribe to Journeyman here: http://www.youtube.com/subscription_c…​

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Watch more episodes of Perspectives on the Pandemic here:

Episode 1: https://dai.ly/x7ubcws​

Episode 2: https://dai.ly/k7af1wKOAvcoA7w5DkZ​

Episode 3: https://youtu.be/VK0Wtjh3HVA​

Episode 4: https://youtu.be/cwPqmLoZA4s​

Episode 5: https://dai.ly/k3l3VyZ2YQv6Zbw5VqE​

Episode 6: https://youtu.be/3f0VRtY9oTs​

Episode 7: https://youtu.be/2JbOvjtnPpE​

Episode 8: https://youtu.be/WlLmt6_w_AM​

Episode 9: https://youtu.be/UIDsKdeFOmQ​

Episode 13: https://youtu.be/UAEAWyfuEWY​

Episode 14: https://youtu.be/J4wIsshE4Q4​

Libby Handros & John Kirby

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And what, in terms of the “pandemic,” is going on in India? One could do worse than to start one’s inquiry here: “How the Unscientific Interpretation of RT-PCR & Rapid Antigen Test Results is Causing Misleading Spikes in Cases & Deaths”

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A link to a .pdf formatted document: How the Unscientific Interpretation of RT-PCR & Rapid Antigen Test Results is Causing Misleading Spikes in Cases & Deaths by Yohan Tengra & Ambar Koiri

“Abstract


“The RT-PCR & RAT tests are currently the main testing method used to diagnose COVID-19. The principle is to collect respiratory cells and to use the RT-PCR (Reverse Transcription – Polymerase Chain Reaction) or RAT (Rapid Antigen Test) technique to detect fragments of the RNA of the virus. The false positive rate is high, between 30%-97%, which can mainly be explained by an incorrect execution of the technique and incorrect interpretation. Previously, these tests were routinely used to diagnose viral upper respiratory tract infections in adults and children, but generally, the test was performed in hospitalized symptomatic patients by an experienced medical team. Currently, all over the world, the public health strategy during this COVID-19 pandemic is based on an early detection of suspicious cases, an early diagnosis of symptomatic patients, and isolation of patients with COVID-19 in order to restrict the outbreak. However, identification of symptoms is currently being skipped, which leads to non-infectious asymptomatic individual being restricted in various ways. The aim of this article is to help healthcare providers to interpret this test correctly in adults and children, & to aid the ICMR in revising its testing guidelines.


“Note – References to backup all the data presented below will be put in blue brackets at the end of every statement and numbered accordingly. You can find the related number at the bottom of the document that will have the relevant link next to it“

Read the document =====> How the Unscientific Interpretation of RT-PCR & Rapid Antigen Test Results is Causing Misleading Spikes in Cases & Deaths

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SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2 — Yuyang Lei et al. | Circulation Research

09 Sunday May 2021

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Source: Circulation Research

SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2

Yuyang Lei, Jiao Zhang, Cara R. Schiavon, Ming He, Lili Chen, Hui Shen, Yichi Zhang, Qian Yin, Yoshitake Cho, Leonardo Andrade, Gerald S. Shadel, Mark Hepokoski, Ting Lei, Hongliang Wang, Jin Zhang, Jason X.-J. Yuan, Atul Malhotra, Uri Manor, Shengpeng Wang, Zu-Yi Yuan, John Y-J. ShyySee fewer authors 

Originally published 31 Mar 2021

https://doi.org/10.1161/CIRCRESAHA.121.318902

Circulation Research. 2021;128:1323–1326

Meet the First Author, see p 1239

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection relies on the binding of S protein (Spike glycoprotein) to ACE (angiotensin-converting enzyme) 2 in the host cells. Vascular endothelium can be infected by SARS-CoV-2,1 which triggers mitochondrial reactive oxygen species production and glycolytic shift.2 Paradoxically, ACE2 is protective in the cardiovascular system, and SARS-CoV-1 S protein promotes lung injury by decreasing the level of ACE2 in the infected lungs.3 In the current study, we show that S protein alone can damage vascular endothelial cells (ECs) by downregulating ACE2 and consequently inhibiting mitochondrial function.

We administered a pseudovirus expressing S protein (Pseu-Spike) to Syrian hamsters intratracheally. Lung damage was apparent in animals receiving Pseu-Spike, revealed by thickening of the alveolar septa and increased infiltration of mononuclear cells (Figure [A]). AMPK (AMP-activated protein kinase) phosphorylates ACE2 Ser-680, MDM2 (murine double minute 2) ubiquitinates ACE2 Lys-788, and crosstalk between AMPK and MDM2 determines the ACE2 level.4 In the damaged lungs, levels of pAMPK (phospho-AMPK), pACE2 (phospho-ACE2), and ACE2 decreased but those of MDM2 increased (Figure [B], i). Furthermore, complementary increased and decreased phosphorylation of eNOS (endothelial NO synthase) Thr-494 and Ser-1176 indicated impaired eNOS activity. These changes of pACE2, ACE2, MDM2 expression, and AMPK activity in endothelium were recapitulated by in vitro experiments using pulmonary arterial ECs infected with Pseu-Spike which was rescued by treatment with N-acetyl-L-cysteine, a reactive oxygen species inhibitor (Figure [B], ii).

Figure.
Figure. SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) Spike protein exacerbates endothelial cell (EC) function via ACE (angiotensin-converting enzyme) 2 downregulation and mitochondrial impairment.A, Representative H&E histopathology of lung specimens from 8- to 12 wk-old male Syrian hamsters 5-day post administration of pseudovirus overexpressing Spike protein (Pseu-Spike) or mock virus in control group (n=3 mice per group, 1×108 PFU). Thickened alveolar septa (red arrowhead) and mononuclear cell (red arrow). Scale bar=20 μm. B, Pseu-Spike (n=4) or mock virus (n=4)–infected hamster lungs were subjected to Western blot analysis for pAMPK (phospho-AMPK) T172, AMPK, pACE2 (phospho angiotensin-converting enzyme) S680, ACE 2, MDM2, peNOS S1176, peNOS T494, eNOS (endothelial NO synthase), and β-actin (B, i). Human pulmonary arterial EC (PAECs) were infected with Pseu-Spike or mock virus for 24 h with or without N-acetyl-L-cysteine (NAC; 5 mmol/L) pretreatment for 2 h. The protein extracts were analyzed by Western blot using antibodies against proteins as indicated (n=4; B, ii). C, Representative confocal images of mitochondrial morphology of ECs treated with human recombinant S1 protein or IgG (4 μg/mL) for 24 h (C, i) or infected with human adenovirus ACE2 S680D (ACE2-D) or ACE2 S680L (ACE2-L; 10 MOI) for 48 h (C, ii). Mitochondria were visualized using TOM20 antibody (n=4, 50 cells counted for each replicate). Scale bar=2.5 μm. Tubular: the majority of mitochondria in ECs was >10 μm in length; Intermediate: the mitochondria were <≈10 μm; Fragment: the majority of mitochondria were spherical (no clear length or width). D, Measurement of oxygen consumption rate (OCR, D, i and iii) and extracellular acidification rate (ECAR, D, ii and iv) in ECs infected with ACE2-D vs ACE2-L (10 MOI) for 48 h (n=3) or treated with IgG vs S1 protein (4 μg/mL) for 24 h (n=3). E, Real-time quantitative polymerase chain reaction analysis of the indicated mRNA levels in lung ECs from ACE2-D (n=4) and ACE2-L (n=4) knock-in mice. Eight-week-old ACE2-D and ACE2-L male mice with C57BL/6 background were used. F, Dose-response curves of acetylcholine (ACh, left)- and sodium nitroprusside (SNP, right)–mediated relaxation on the tension of phenylephrine (1 μmol/L) precontracted intrapulmonary artery stripes from Pseu-Spike-(ACh n=8, SNP n=5) or mock (ACh n=6, SNP n=5) virus–infected Syrian hamsters (1×108 PFU; F, i) and ACE2-D (n=6) or ACE2-L (n=5) mice (F, ii). The animal experiments were approved by the ethical committee of Xi’an Jiaotong University. 2-DG indicates 2-Deoxy-D-glucose; ACE2-D, a phospho-mimetic ACE2 with increased stability; ACE2-L, a dephospho-mimetic ACE2 with decreased stability; AMPK, AMP-activated protein kinase; AA/R, antimycin A&Rotenone; ENO2, enolase 2; FCCP, carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone; H&E, Hematoxylin and Eosin; HK2, hexokinase 2; HO1, heme oxygenase-1; MDM2, murine double minute 2; MOI, multiplicity of infection; NRF1, nuclear respiratory factor 1; peNOS, phospho-eNOS; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; Resp, respiration; and TFAM, transcription factor A, mitochondrial.

We next studied the impact of S protein on mitochondrial function. Confocal images of ECs treated with S1 protein revealed increased mitochondrial fragmentation, indicating altered mitochondrial dynamics (Figure [C], i). To examine whether these mitochondrial changes were due, in part, to the decreased amount of ACE2, we overexpressed ACE2 S680D (ACE2-D, a phospho-mimetic ACE2 with increased stability) or S680L (ACE2-L, a dephospho-mimetic with decreased stability)4 in ECs. As shown in Figure [C], ii, ECs with ACE2-L had a higher number of fragmented mitochondria when compared to those with ACE2-D. Performing oxygen consumption rate and extracellular acidification rate assays, we found that ECs overexpressing ACE2-L had reduced basal mitochondrial respiration, ATP production, and maximal respiration compared to ECs overexpressing ACE2-D (Figure [D], i). Moreover, ACE2-L overexpression caused increased basal acidification rate, glucose-induced glycolysis, maximal glycolytic capacity, and glycolytic reserve (Figure [D], ii). Also, ECs incubated with S1 protein had attenuated mitochondrial function but increased glycolysis, when compared with control cells treated with IgG (Figure [D], iii and iv). We also compared the expressions of mitochondria- and glycolysis-related genes in lung ECs isolated from ACE2-D or ACE2-L knock-in mice.4 Shown in Figure [E], the mRNA levels of NRF1, HO1, and TFAM (mitochondria biogenesis-related genes) were increased, whereas those of HK2, PFKFB3, and ENO2 (glycolysis-related genes) were decreased in lung ECs in ACE2-D mice, as compared to those in ACE2-L mice.

SARS-CoV-2 infection induces EC inflammation, leading to endotheliitis.1,5 Because S protein decreased ACE2 level and impaired NO bioavailability, we examined whether S protein entry is indispensable for dysfunctional endothelium. As shown in Figure [F], i, the endothelium-dependent vasodilation induced by acetylcholine was impaired in pulmonary arteries isolated from Pseu-Spike-administered hamsters, whereas the endothelium-independent vasodilation induced by sodium nitroprusside was not affected. We also compared the acetylcholine- and sodium nitroprusside–induced vasodilation of pulmonary vessels from ACE2-D or ACE2-L mice. As anticipated, acetylcholine-induced vasodilation was hindered in pulmonary arteries isolated from ACE2-L mice in comparison to ACE2-D mice (Figure [F], ii). There was, however, little difference in sodium nitroprusside–induced vasodilation between ACE2-D and ACE-L animals.

Although the use of a noninfectious pseudovirus is a limitation to this study, our data reveals that S protein alone can damage endothelium, manifested by impaired mitochondrial function and eNOS activity but increased glycolysis. It appears that S protein in ECs increases redox stress which may lead to AMPK deactivation, MDM2 upregulation, and ultimately ACE2 destabilization.4 Although these findings need to be confirmed with the SARS-CoV-2 virus in the future study, it seems paradoxical that ACE2 reduction by S protein would decrease the virus infectivity, thereby protecting endothelium. However, a dysregulated renin-angiotensin system due to ACE2 reduction may exacerbate endothelial dysfunction, leading to endotheliitis. Collectively, our results suggest that the S protein-exerted EC damage overrides the decreased virus infectivity. This conclusion suggests that vaccination-generated antibody and/or exogenous antibody against S protein not only protects the host from SARS-CoV-2 infectivity but also inhibits S protein-imposed endothelial injury.

Nonstandard Abbreviation and Acronyms

ACEangiotensin-converting enzyme
ECsendothelial cells
eNOSendothelial NO synthase
pACE2phospho-ACE2
pAMPKphospho-AMPK
S proteinSpike glycoprotein

Data Availability

The data that support the findings of this study, including statistical analyses and reagents used, are available from the corresponding author upon request.

Sources of Funding

This work was supported in part by grants from the National Natural Science Foundation of China (NSFC) grants 81870220 (S. Wang), 81800328 (J.Z.), 81941005 (Z.-Y. Yuan); Shaanxi Natural Science Fund S2020-JC-JQ-0239 (S. Wang); The National Key Research and Development Program (Grant No. 2018YFC1311500; Z.-Y. Yuan); the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University (Grant No. XJTU1AF-CRF-2016-004; Z.-Y. Yuan); Xi’an Jiaotong University Financial support.

Disclosures None.

Footnotes

*Y. Lei and J. Zhang contributed equally.

†U. Manor, S. Wang, Z.-Y. Yuan, and J.Y.-J. Shyy contributed equally as senior authors.

For Sources of Funding and Disclosures, see page 1324.Correspondence to: John Y-J. Shyy, PhD, Division of Cardiology, Department of Medicine, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093, Email jshyy@health.ucsd.eduZu-Yi Yuan, MD, PhD, Department of Cardiology, First Affiliated Hospital of Xi’an Jiaotong University, 277 Yanta W Rd, Xi’an 710061, China, Email zuyiyuan@mail.xjtu.edu.cn

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