Covid-19: The virus attacks mitochondria, continuing an ancient battle that began in the primordial soup
Kingston (Canada), (The Conversation): Viruses and bacteria have a very long history. Because viruses can’t reproduce without a host, they’ve been attacking bacteria for millions of years. Some of those bacteria eventually became mitochondria, synergistically adapting to life within eukaryotic cells (cells that have a nucleus containing chromosomes).
Ultimately, mitochondria became the powerhouses within all human cells.
Fast-forward to the rise of novel coronaviruses like SARS-CoV-2, and the global spread of COVID-19. Approximately five per cent of people infected with SARS-CoV-2 suffer respiratory failure (low blood oxygen) requiring hospitalization. In Canada about 1.1 per cent of infected patients (almost 46,000 people) have died.
This is the story of how a team, assembled during the pandemic, recognized the mechanism by which these viruses were causing lung injury and lowering oxygen levels in patients: It is a throwback to the primitive war between viruses and bacteria — more specifically, between this novel virus and the evolutionary offspring of bacteria, our mitochondria.
SARS-CoV-2 is the third novel coronavirus to cause human outbreaks in the 21st century, following SARS-CoV in 2003 and MERS-CoV in 2012. We need to better understand how coronaviruses cause lung injury to prepare for the next pandemic.
How COVID-19 affects lungs
People with severe COVID-19 pneumonia often arrive at the hospital with unusually low oxygen levels. They have two unusual features distinct from patients with other types of pneumonia:
First, they suffer widespread injury to their lower airway (the alveoli, which is where oxygen is taken up).
Second, they shunt blood to unventilated areas of the lung, which is called ventilation-perfusion mismatch. This means blood is going to parts of the lung where it won’t get sufficiently oxygenated.
Together, these abnormalities lower blood oxygen. However, the cause of these abnormalities was unknown. In 2020, our team of 20 researchers at three Canadian universities set about to unravel this mystery. We proposed that SARS-CoV-2 worsened COVID-19 pneumonia by targeting mitochondria in airway epithelial cells (the cells that line the airways) and pulmonary artery smooth muscle cells.
We already knew that mitochondria are not just the powerhouse of the cell, but also its main consumers and sensors of oxygen. Mitochondria control the process of programmed cell death (called apoptosis), and they regulate the distribution of blood flow in the lung by a mechanism called hypoxic pulmonary vasoconstriction.
This mechanism has an important function. It directs blood away from areas of pneumonia to better ventilated lobes of the lung, which optimizes oxygen-uptake. By damaging the mitochondria in the smooth muscle cells of the pulmonary artery, the virus allows blood flow to continue into areas of pneumonia, which also lowers oxygen levels.
It appeared plausible that SARS-CoV-2 was damaging mitochondria. The results of this damage — an increase in apoptosis in airway epithelial cells, and loss of hypoxic pulmonary vasoconstriction — were making lung injury and hypoxemia (low blood oxygen) worse.
Our discovery, published in Redox Biology, explains how SARS-CoV-2, the coronavirus that causes COVID-19 pneumonia, reduces blood oxygen levels.
We show that SARS-CoV-2 kills airway epithelial cells by damaging their mitochondria. This results in fluid accumulation in the lower airways, interfering with oxygen uptake. We also show that SARS-CoV-2 damages mitochondria in the pulmonary artery smooth muscle cells, which inhibits hypoxic pulmonary vasoconstriction and lowers oxygen levels.
Coronaviruses damage mitochondria in two ways: by regulating mitochondria-related gene expression, and by direct protein-protein interactions. When SARS-CoV-2 infects a cell, it hijacks the host’s protein synthesis machinery to make new virus copies. However, these viral proteins also target host proteins, causing them to malfunction. We soon learned that many of the host cellular proteins targeted by SARS-CoV-2 were in the mitochondria.
Viral proteins fragment the mitochondria, depriving cells of energy and interfering with their oxygen-sensing capability. The viral attack on mitochondria starts within hours of infection, turning on genes that break the mitochondria into pieces (called mitochondrial fission) and make their membranes leaky (an early step in apoptosis called mitochondrial depolarization).
In our experiments, we didn’t need to use a replicating virus to damage the mitochondria — simply introducing single SARS-CoV-2 proteins was enough to cause these adverse effects. This mitochondrial damage also occurred with other coronaviruses that we studied.
We are now developing drugs that may one day counteract COVID-19 by blocking mitochondrial fission and apoptosis, or by preserving hypoxic pulmonary vasoconstriction. Our drug discovery efforts have already enabled us to identify a promising mitochondrial fission inhibitor, called Drpitor1a.
Our team’s infectious diseases expert, Gerald Evans, notes that this discovery also has the potential to help us understand Long COVID. “The predominant features of that condition — fatigue and neurologic dysfunction — could be due to the lingering effects of mitochondrial damage caused by SARS-CoV-2 infection,” he explains.
The ongoing evolutionary battle
This research also has an interesting evolutionary angle. Considering that mitochondria were once bacteria, before being adopted by cells back in the primordial soup, our findings reveal an Alien versus Predator scenario in which viruses are attacking “bacteria.”
Bacteria are regularly attacked by viruses, called bacteriophages, that need a host to replicate in. The bacteria in turn fight back, using an ancient form of immune system called the CRISPR-cas system, that chops up the viruses’ genetic material. Humans have recently exploited this CRISPR-cas system for a Nobel Prize-winning gene editing discovery.
The ongoing competition between bacteria and viruses is a very old one; and recall that our mitochondria were once bacteria. So perhaps it’s not surprising at all that SARS-CoV-2 attacks our mitochondria as part of the COVID-19 syndrome.
The original team members on this project are heart and lung researchers with expertise in mitochondrial biology. In early 2020 we pivoted to apply that in another field — virology — in an effort to make a small contribution to the COVID-19 puzzle.
The diverse team we put together also brought expertise in mitochondrial biology, cardiopulmonary physiology, SARS-CoV-2, transcriptomics, synthetic chemistry, molecular imaging and infectious diseases.
Our discovery owes a lot to our virology collaborators. Early in the pandemic, University of Toronto virologist Gary Levy offered us a mouse coronavirus (MHV-1) to work with, which we used to make a model of COVID-19 pneumonia. Che Colpitts, a virologist at Queen’s University, helped us study the mitochondrial injury caused by another human beta coronavirus, HCoV-OC43.
Finally, Arinjay Banerjee and his expert SARS-CoV-2 virology team at Vaccine and Infectious Disease Organization (VIDO) in Saskatoon performed key studies of human SARS-CoV-2 in airway epithelial cells. VIDO is one of the few Canadian centres equipped to handle the highly infectious SARS-CoV-2 virus.
Our team’s super-resolution microscopy expert, Jeff Mewburn, notes the specific challenges the team had to contend with.
“Having to follow numerous and extensive COVID-19 protocols, they were still able to exhibit incredible flexibility to retool and refocus our laboratory specifically on the study of coronavirus infection and its effects on cellular/mitochondrial functions, so very relevant to our global situation,” he said.
Our discovery will hopefully be translated into new medicines to counter future pandemics.
COVID-19 is normally associated with the lungs and respiratory system. Most people think of symptoms such as coughing and shortness of breath when they think of COVID-19. However, COVID-19 can impact the entire body and cause a wide range of symptoms and complications.
Strokes, a vascular system condition, are among the most common complications for people hospitalized with severe COVID-19. This led many researchers to question if COVID-19 is a vascular disease with respiratory symptoms.
Like so many things about COVID-19, we’re still not 100 percent certain of the answer to that question. However, the latest research shows that the vascular symptoms of COVID-19 are caused by inflammation and not COVID-19.
This means COVID-19 is still considered a respiratory disease, but it can have serious effects on the vascular system. Read on to learn more.
Medical professionals and researchers have been studying the link between COVID-19 and vascular symptoms since the beginning of the pandemic. They have learned that people with severe COVID-19 are at a risk of strokes, blood clots, and other vascular complications.
These observations led to several hypotheses that COVID-19 was a vascular disease with respiratory symptoms and not a respiratory disease.
Studies in 2020Trusted Source and 2021 supported this theory. These studies concluded that although people with mild to moderate COVID-19 only had respiratory symptoms, COVID-19 was primarily a vascular disease. However, additional studies published later in 2021Trusted Source and into 2022Trusted Source have contraindicated these findings. New studies indicate that COVID-19 doesn’t attack the vascular system at all.
Instead, these studies found that strokes and other vascular complications occur when infected respiratory cells cause extreme inflammation in other parts of your body.
Many people who are hospitalized for COVID-19 are at increased risk of vascular complications. Knowing that these complications are part of an inflammatory immune system response can help doctors lower the risk of stroke and other serious vascular complications.
For example, people with SARS-CoV-2 infections who are at risk of vascular complications may be given blood thinners to help lower their risk. Doctors, medical researchers, and other professionals might also look for ways to lower inflammation while still helping the body fight COVID-19.
Understanding how COVID-19 affects the vascular system can also help researchers identify people who are most at risk of vascular complications, leading to targeted treatments and better outcomes.
Like many things related to COVID-19, more research about this connection still needs to be done.
COVID-19 is known to have both short-term and long-term symptoms and complications. Some of these symptoms are respiratory and sensory. For instance, you might’ve read articles about people who lost their sense of smell for months following the development of COVID-19.
There are also long-term complications and symptoms associated with the vascular symptom. Not everyone will have these symptoms, but studying them has been an important part of researchers understanding how COVID-19 affects the vascular system.
Long-term vascular complications of COVID-19 include:
In studies, heart failure and arrhythmias were the most common vascular complications of COVID-19. However, the data on complications from COVID-19 is still very new.
People who have recovered from COVID-19 have only been observed for a year or two. What we know about vascular and other complications might change in the years to come as the first people who recovered from COVID-19 are observed for longer.
Additionally, new COVID-19 treatments might drastically change what complications look like for future SARS-CoV-2 infections.
Yes. COVID-19 can cause serious organ damage. Your lungs, liver, kidneys, brain, and heart can all be damaged by COVID-19.
Which organ is most often affected by COVID-19?
The lungs are the organs most affected by COVID-19. COVID-19 can irritate the lining of your lungs, cause inflammation in your lungs, cause your lungs to fill with fluid, and can cause damage to the lining of your lungs.
Not everyone who gets COVID-19 will experience lung damage. For many people, COVID-19 presents as a mild respiratory infection, but severe COVID-19 can lead to organ damage and even death.
Does COVID-19 damage your heart?
COVID-19 can cause damage to multiple organs, including damage to your heart. People who’ve recovered from severe COVID-19 are at an increased risk of heart complications. This indicates a strong link between COVID-19 and heart health.
Additionally, there’s evidence that people who already have heart conditions are at risk of more serious symptoms if they do develop COVID-19.
Since the early days of the pandemic, researchers have noticed that a large number of people hospitalized with severe COVID-19 had strokes, blood clots, and other vascular complications. This led to theories and studies about the link between COVID-19 and the vascular system.
Currently, researchers believe that the immune system attacks infected respiratory cells as they travel through the rest of the body. This response can sometimes cause significant inflammation, damage the lining of your blood vessels, and lead to blood clots.
More research still needs to be done on this topic, but what we now know is already helping doctors lower the risk of stroke and clots for those hospitalized with severe COVID-19.
Here’s what research says about how long COVID lung disease, mental health, brain fog, and more may impact people who are immunocompromised.
Throughout the pandemic, experts have continued to learn more about long COVID (also known as long-haul COVID and post-COVID conditions) — and what it means for risk of lung disease, heart disease, mental health conditions, cognitive issues, and more.
If you’re immunocompromised and at risk for severe COVID-19, you’ve likely already been taking every step possible to lower your chances of getting infected. However, if you do contract COVID-19, it’s important to know how it affects your risk of other health conditions so you can work with your doctor to monitor symptoms.
It’s not clear if being immunocompromised alone makes you more likely to experience long COVID. However, you may be at greater risk simply due to your higher likelihood of developing severe COVID-19.
“I have not seen data to suggest confirming that immunocompromised patients are more likely to develop long COVID than patients who are not immunocompromised,” says Samoon Ahmad, MD, clinical professor of psychiatry at NYU Grossman School of Medicine. “That said, it’s clear that immunocompromised patients are more likely to develop severe COVID if they get it — and research suggests that people who have severe COVID are more likely to develop long COVID.”
However, more research is needed to confirm this link.
Meanwhile, factors such as older age, being female, and hospitalization at symptom onset have been found to be significantly associated with an increased risk of developing persistent symptoms, per a July 2021 review in the Journal of the Royal Society of Medicine.
Needing oxygen therapy, pre-existing hypertension, and chronic lung conditions were also highlighted in the study as being major factors of long-term symptoms.
So what does this mean for your long-term health and risk of chronic disease? Here are potential complications of long COVID you should know about as an immunocompromised patient.
Long COVID and Lung Disease
COVID-19 can cause both short-term and long-term complications to your lungs, but the way in which it does has changed over the course of the pandemic.
You may remember that at the beginning of the pandemic, many people experienced COVID-related pneumonia. This resulted in oxygen levels dropping, feelings of breathlessness, and eventually hospitalization. That’s because the earlier variants had a tendency to infect the lung tissue.
“With those variants, we saw a lot more scarring happening to the lungs,” says Panagis Galiatsatos, MD, assistant professor of medicine at Johns Hopkins Medicine. “For most people, the scarring kind of came and left. For others, it remained permanent — and then there’s a small subgroup where the scarring actually never ‘shut off’ and they developed post-COVID-19 fibrosis.”
The newer variants, particularly Omicron, are much more involved in the airways (the tubes that lead to the lungs). That results in much more coughing during infection, but less of a drop in oxygen levels.
A recent University of Iowa Health Care study of 100 participants revealed that air trapping persisted in eight out of nine participants imaged more than 200 days after COVID-19 diagnosis. Air trapping is a condition in which people cannot empty their lungs when they breathe out, which is indicative of small airways disease, and it leads to side effects such as shortness of breath.
The researchers found that the percentage of lung affected by air trapping was similar across patients, regardless of how severe their symptoms were.
What’s more, with the more recent variants, the most common complication patients have post-COVID is a post-viral cough that takes three to six months to go away. During this time, your lungs are essentially trying to “cough out” the affected cells.
“The lungs are going to clear out the cells that were invaded,” says Dr. Galiatsatos. “This cough is very normal. We can suppress it if you need, but this is your lungs’ way of getting things out.”
That said, if you’re immunocompromised, you could experience abnormal healing. Touch base with your physician when you’re recovering from COVID-19 so they can monitor your cough or any other lingering symptoms. While a doctor may normally look further into a cough that lasts longer than six months, if you’re immunocompromised, that timeframe may shorten.
“If an immunosuppressed patient has a cough even just a month after COVID-19, I scan their chest and make sure things are going OK,” says Dr. Galiatsatos. Certain patients, like those who are older or have preexisting pulmonary conditions like asthma, are more vulnerable to developing ongoing pulmonary symptoms.
In another recent study published in Radiology, researchers assessed lung abnormalities in 91 participants (mean age of 59 years) one year after they had COVID-19 pneumonia. At one year, CT scan abnormalities were found in 54 percent of the participants — 4 percent of which had received outpatient care only, 51 percent of which were treated on a general hospital ward, and 45 percent of which had received intensive care unit treatment.
What’s more, 63 percent of participants with abnormalities did not show additional improvements after six months. Being older than 60 years, critical COVID-19 severity, and being male were associated with persistent CT abnormalities at one year.
Long COVID and Diabetes
There may also be a link between long COVID and the development of type 2 diabetes. In a May 2022 study in The Lancet Diabetes & Endocrinology, researchers used the national databases of the U.S. Department of Veterans Affairs to analyze data from more than 8.5 million participants before and during the pandemic.
They found that people who had been infected with COVID-19 were about 40 percent more likely to develop diabetes up to a year later than those in a control group.
Almost all cases were type 2 diabetes, in which the body doesn’t produce enough insulin or becomes resistant to it. Patients who were hospitalized or admitted to intensive care had roughly triple the risk compared to control participants who did not have COVID-19 — but even those with mild infections and no previous diabetes risk factors had a higher chance of developing the condition.
“The mechanism(s) underpinning the association between COVID-19 and risk of diabetes are not entirely clear,” note the researchers.
However, it’s clear that prevention and monitoring for diabetes should be part of the post-COVID strategies, particularly for those who experienced severe COVID-19.
“Current evidence suggests that diabetes is a facet of the multifaceted long COVID syndrome,” add the researchers. “Post-acute care strategies of people with COVID-19 should include identification and management of diabetes.” (Acute COVID-19 is the stage of infection that typically lasts four weeks from the onset of symptoms, per a review in Nature Medicine.)
Long COVID and Mental Health
Long COVID has also been linked to a variety of mental health and cognitive issues, including:
Depression & Anxiety
COVID-19 may increase your chances of experiencing depression or anxiety. An observational follow-up study in six European countries published in The Lancet Public Health found that COVID-19 survivors who were bedridden for more than seven days had a persistently higher risk for depression (61%) and anxiety (43%) than uninfected participants throughout the study period.
A 2021 study published in Cardiovascular Diabetology suggests that long COVID is primarily caused by microclots that starve different cells of oxygen. These microclots form around trapped inflammatory markers.
“I think this mild hypoxia [deprivation of oxygen in tissues] can lead to inflammation and activation of microglia,” says Dr. Ahmad. “These microglia are cells in the brain that release inflammatory signals when activated, which then leads to neuroinflammation.” This could potentially explain the pathology of long COVID and associated issues like anxiety.
Continuing to avoid crowds or choosing to work from home doesn’t necessarily mean you have clinical anxiety. However, anxiety does become a clinical problem when it disrupts your life to the point of you avoiding social, occupational, or academic obligations. For instance, if you feel too anxious to pick up phone calls from friends or family.
“If your anxiety is so severe that it makes it interferes with your ability to live your life, then you definitely want to speak to a doctor,” says Dr. Ahmad. “You may also want to tell your doctor about your anxiety if it is part of a larger cluster of symptoms — including shortness of breath, fatigue, or if you are slow to heal from small cuts or bruises.”
It’s important to stay in touch with your doctor and keep a symptom journal if necessary.
“Immunocompromised patients should know that anxiety is one of the symptoms of long COVID and that it can be exacerbated by other symptoms,” says Dr. Ahmad. “For example, many patients with long COVID report sleep problems (“COVID-somnia”). When you don’t sleep well, this can make your anxiety worse.”
Sleep disturbances are estimated to affect up to 50 to 75 percent of COVID-19 patients, per a 2021 review in the Journal of Personalized Medicine. And of course, if you’re living with another underlying condition, symptoms like insomnia or pain may be commonplace for you.
“A similar thing can be said of several other common long COVID symptoms, including fatigue and shortness of breath,” says Dr. Ahmad. “When a patient feels severe shortness of breath, this may even trigger a panic attack.”
Meanwhile, it’s normal to feel sad sometimes (especially during a global pandemic), but if you’re persistently sad, anxious, or in an “empty” mood, it could be a symptom of depression, per the National Institute of Mental Health.
Difficulty with concentration and memory have also been attributed to long COVID. In fact, brain fog — the feeling of slow or sluggish thinking — occurs in an estimated 22 to 32 percent of patients who recover from COVID-19, per Harvard Medical School.
In a recent study in the journal Nature, researchers analyzed brain changes in 785 participants ages 51-81 whose brains were scanned twice (including 401 people who contracted COVID-19 between their two scans). They found evidence that COVID-19 can cause the brain to shrink by reducing grey matter in regions that control emotion and memory.
“The participants who were infected with SARS-CoV-2 also showed on average a greater cognitive decline between the two time points,” note the researchers.
The effects were even seen in those who were not hospitalized with COVID-19. More research is needed to determine if this impact could be partially reversed or if it will persist in the long-term.
Meanwhile, a January 2022 study published in Brain Communications suggests that some people may have problems with memory and attention after recovering from a mild case of COVID-19, even if they don’t realize it.
Testing showed that performance on tasks involving attention and memory were poorer in participants who had COVID-19 compared to those that didn’t. However, in this study, both of the effects seemed to improve within six to nine months.
“My doctor said I was COVID long-hauler after my symptoms continued from June of 2020. Recently, I began noticing brain fog, a symptom I haven’t experienced in quite some time,” says JP Summers, an Advocacy Fellow at the Global Health Living Foundation who lives with migraine, fibromyalgia, rheumatoid arthritis, and heart disease. “My mind goes completely blank. A heavy cloud of confusion sets in and I feel lost on where I am or what I was doing at that moment. It is both incredibly frustrating and terrifying, especially when it happens in a public place.” JP has been actively tracking her symptoms to discuss with her cardiologist at her next appointment.
Long COVID and Heart Disease
Heart health has been a major focus during the COVID-19 pandemic, with several cardiovascular effects appearing to be associated with long COVID.
In a February 2022 study published in Nature Medicine, researchers analyzed 154,000 U.S. veterans (plus over 10 million patients who served as historical and contemporary control groups). They found that in the year after recovering from COVID-19, patients had increased risks of several cardiovascular issues, including abnormal heart rhythms, heart muscle inflammation, blood clots, strokes, myocardial infarction, and heart failure — even if they weren’t hospitalized with COVID-19.
The risks were evident regardless of age, race, sex, and other cardiovascular risk factors such as obesity, hypertension, diabetes, chronic kidney disease, and hyperlipidemia (high levels of fat particles in the blood). The risks were also evident in those who did not have any cardiovascular disease before exposure to COVID-19, showing that these risks may manifest even in those at low risk of heart disease.
“Our results provide evidence that the risk and 1-year burden of cardiovascular disease in survivors of acute COVID-19 are substantial,” note the researchers. “Care pathways of those surviving the acute episode of COVID-19 should include attention to cardiovascular health and disease.”
Your heart and lungs work together to deliver oxygen-rich blood to your body, but COVID-19 can disrupt both. COVID-19 can cause lung damage, keeping oxygen from reaching the heart muscle, meaning your heart has to work harder to get oxygen to other tissues in the body, per the University of Maryland Medical System.
COVID-19 can also cause an excess of inflammation, which may further damage the heart and affect the electrical signals that help it beat properly. This can lead to abnormal heart rhythm or exacerbate an existing rhythm problem.
Work with your doctor to monitor your heart health and practice a heart-healthy lifestyle. This includes staying active, eating a healthy diet, managing your stress, and quitting if you’re a smoker.
The bottom line: Although research is showing that long-term complications from COVID-19 are prevalent.
Peer Reviewed Medical Papers Submitted To Various Medical Journals, Evidencing A Multitude Of Adverse Events In Covid-19 Vaccine Recipients.
The list includes studies published as of January 20, 2022 concerning the potential adverse reaction from COVID-19 vaccines, such as myocarditis, thrombosis, thrombocytopenia, vasculitis, cardiac, Bell’s Palsy, immune-mediated disease, and many more.
Occurrence of acute infarct-like myocarditis after COVID-19 vaccination: just an accidental coincidence or rather a vaccination-associated autoimmune myocarditis?: https://pubmed.ncbi.nlm.nih.gov/34333695/
This list is not meant to be all inclusive of all peer-reviewed potential harms from mRNA vaccines. To access any of the 1,000 Vaccine Harms published in Medical journals Click The Link Below:
Authors.Maximilian Ackermann, M.D., Stijn E. Verleden, Ph.D., Mark Kuehnel, Ph.D., Axel Haverich, M.D., Tobias Welte, M.D., Florian Laenger, M.D., Arno Vanstapel, Ph.D., Christopher Werlein, M.D., Helge Stark, Ph.D., Alexandar Tzankov, M.D., William W. Li, M.D., Vincent W. Li, M.D., et al.
July 9, 2020 N Engl J Med 2020; 383:120-128 DOI: 10.1056/NEJMoa2015432
Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (Covid-19) pandemic. Despite widespread interest in the pathophysiology of the disease, relatively little is known about the associated morphologic and molecular changes in the peripheral lung of patients who die from Covid-19.
We examined 7 lungs obtained during autopsy from patients who died from Covid-19 and compared them with 7 lungs obtained during autopsy from patients who died from acute respiratory distress syndrome (ARDS) secondary to influenza A(H1N1) infection and 10 age-matched, uninfected control lungs. The lungs were studied with the use of seven-color immunohistochemical analysis, micro–computed tomographic imaging, scanning electron microscopy, corrosion casting, and direct multiplexed measurement of gene expression.
In patients who died from Covid-19–associated or influenza-associated respiratory failure, the histologic pattern in the peripheral lung was diffuse alveolar damage with perivascular T-cell infiltration. The lungs from patients with Covid-19 also showed distinctive vascular features, consisting of severe endothelial injury associated with the presence of intracellular virus and disrupted cell membranes. Histologic analysis of pulmonary vessels in patients with Covid-19 showed widespread thrombosis with microangiopathy. Alveolar capillary microthrombi were 9 times as prevalent in patients with Covid-19 as in patients with influenza (P<0.001). In lungs from patients with Covid-19, the amount of new vessel growth — predominantly through a mechanism of intussusceptive angiogenesis — was 2.7 times as high as that in the lungs from patients with influenza (P<0.001).
In our small series, vascular angiogenesis distinguished the pulmonary pathobiology of Covid-19 from that of equally severe influenza virus infection. The universality and clinical implications of our observations require further research to define. (Funded by the National Institutes of Health and others.)
Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans is associated with a broad spectrum of clinical respiratory syndromes, ranging from mild upper airway symptoms to progressive life-threatening viral pneumonia.1,2 Clinically, patients with severe coronavirus disease 2019 (Covid-19) have labored breathing and progressive hypoxemia and often receive mechanical ventilatory support. Radiographically, peripheral lung ground-glass opacities on computed tomographic (CT) imaging of the chest fulfill the Berlin criteria for acute respiratory distress syndrome (ARDS).3,4 Histologically, the hallmark of the early phase of ARDS is diffuse alveolar damage with edema, hemorrhage, and intraalveolar fibrin deposition, as described by Katzenstein et al.5 Diffuse alveolar damage is a nonspecific finding, since it may have noninfectious or infectious causes, including Middle East respiratory syndrome coronavirus (MERS-CoV),6 SARS-CoV,7 SARS-CoV-2,8-10 and influenza viruses.11
Among the distinctive features of Covid-19 are the vascular changes associated with the disease. With respect to diffuse alveolar damage in SARS-CoV7 and SARS-CoV-2 infection,8,12 the formation of fibrin thrombi has been observed anecdotally but not studied systematically. Clinically, many patients have elevated d-dimer levels, as well as cutaneous changes in their extremities suggesting thrombotic microangiopathy.13 Diffuse intravascular coagulation and large-vessel thrombosis have been linked to multisystem organ failure.14-16 Peripheral pulmonary vascular changes are less well characterized; however, vasculopathy in the gas-exchange networks, depending on its effect on the matching of ventilation and perfusion that results, could potentially contribute to hypoxemia and the effects of posture (e.g., prone positioning) on oxygenation.17
Despite previous experience with SARS-CoV18 and early experience with SARS-CoV-2, the morphologic and molecular changes associated with these infections in the peripheral lung are not well documented. Here, we examine the morphologic and molecular features of lungs obtained during autopsy from patients who died from Covid-19, as compared with those of lungs from patients who died from influenza and age-matched, uninfected control lungs.
PATIENT SELECTION AND WORKFLOW
We analyzed pulmonary autopsy specimens from seven patients who died from respiratory failure caused by SARS-CoV-2 infection and compared them with lungs from seven patients who died from pneumonia caused by influenza A virus subtype H1N1 (A[H1N1]) — a strain associated with the 1918 and 2009 influenza pandemics. The lungs from patients with influenza were archived tissue from the 2009 pandemic and were chosen for the best possible match with respect to age, sex, and disease severity from among the autopsies performed at the Hannover Medical School. Ten lungs that had been donated but not used for transplantation served as uninfected control specimens. The Covid-19 group consisted of lungs from two female and five male patients with mean (±SD) ages of 68±9.2 years and 80±11.5 years, respectively (clinical data are provided in Table S1A in the Supplementary Appendix, available with the full text of this article at NEJM.org). The influenza group consisted of lungs from two female and five male patients with mean ages of 62.5±4.9 years and 55.4±10.9 years, respectively. Five of the uninfected lungs were from female donors (mean age, 68.2±6.9 years), and five were from male donors (mean age, 79.2±3.3 years) (clinical data are provided in Table S1B). The study was approved by and conducted according to requirements of the ethics committees at the Hannover Medical School and the University of Leuven. There was no commercial support for this study.
All lungs were comprehensively analyzed with the use of microCT, histopathological, and multiplexed immunohistochemical analysis, transmission and scanning electron microscopy, corrosion casting, and direct multiplexed gene-expression analysis, as described in detail in the Methods section of the Supplementary Appendix.
All comparisons of numeric variables (including those in the gene-expression analysis) were conducted with Student’s t-test familywise error rates due to multiplicity set at 0.05 with the use of the Benjamini–Hochberg method of controlling false discovery rates. Original P values are reported only for the tests that met the criteria for false discovery rates. All confidence intervals have been calculated on the basis of the t-distribution, as well. Additional details are provided in the Methods section of the Supplementary Appendix.
The mean (±SE) weight of the lungs from patients with proven influenza pneumonia was significantly higher than that from patients with proven Covid-19 (2404±560 g vs. 1681±49 g; P=0.04). The mean weight of the uninfected control lungs (1045±91 g) was significantly lower than those in the influenza group (P=0.003) and the Covid-19 group (P<0.001).
Figure 1. Lymphocytic Inflammation in a Lung from a Patient Who Died from Covid-19.
All lung specimens from the Covid-19 group had diffuse alveolar damage with necrosis of alveolar lining cells, pneumocyte type 2 hyperplasia, and linear intraalveolar fibrin deposition (Figure 1). In four of seven cases, the changes were focal, with only mild interstitial edema. The remaining three cases had homogeneous fibrin deposits and marked interstitial edema with early intraalveolar organization. The specimens in the influenza group had florid diffuse alveolar damage with massive interstitial edema and extensive fibrin deposition in all cases. In addition, three specimens in the influenza group had focal organizing and resorptive inflammation (Fig. S2). These changes were reflected in the much higher weight of the lungs from patients with influenza.
Immunohistochemical analysis of angiotensin-converting enzyme 2 (ACE2) expression, measured as mean (±SD) relative counts of ACE2-positive cells per field of view, in uninfected control lungs showed scarce expression of ACE2 in alveolar epithelial cells (0.053±0.03) and capillary endothelial cells (0.066±0.03). In lungs from patients with Covid-19 and lungs from patients with influenza, the relative counts of ACE2-positive cells per field of view were 0.25±0.14 and 0.35±0.15, respectively, for alveolar epithelial cells and 0.49±0.28 and 0.55±0.11, respectively, for endothelial cells. Furthermore, ACE2-positive lymphocytes were not seen in perivascular tissue or in the alveoli of the control lungs but were present in the lungs in the Covid-19 group and the influenza group (relative counts of 0.22±0.18 and 0.15±0.09, respectively). (Details of counting are provided in Table S2.)
In the lungs from patients with Covid-19 and patients with influenza, similar mean (±SD) numbers of CD3-positive T cells were found within a 200-μm radius of precapillary and postcapillary vessel walls in 20 fields of examination per patient (26.2±13.1 for Covid-19 and 14.8±10.8 for influenza). With the same field size used for examination, CD4-positive T cells were more numerous in lungs from patients with Covid-19 than in lungs from patients with influenza (13.6±6.0 vs. 5.8±2.5, P=0.04), whereas CD8-positive T cells were less numerous (5.3±4.3 vs. 11.6±4.9, P=0.008). Neutrophils (CD15 positive) were significantly less numerous adjacent to the alveolar epithelial lining in the Covid-19 group than in the influenza group (0.4±0.5 vs. 4.8±5.2, P=0.002).
A multiplexed analysis of inflammation-related gene expression examining 249 genes from the nCounter Inflammation Panel (NanoString Technologies) revealed similarities and differences between the specimens in the Covid-19 group and those in the influenza group. A total of 79 inflammation-related genes were differentially regulated only in specimens from patients with Covid-19, whereas 2 genes were differentially regulated only in specimens from patients with influenza; a shared expression pattern was found for 7 genes (Fig. S1).
THROMBOSIS AND MICROANGIOPATHY
Figure 2. Microthrombi in the Interalveolar Septa of a Lung from a Patient Who Died from Covid-19.
The pulmonary vasculature of the lungs in the Covid-19 group and the influenza group was analyzed with hematoxylin–eosin, trichrome, and immunohistochemical staining (as described in the Methods section of the Supplementary Appendix). Analysis of precapillary vessels showed that in four of the seven lungs from patients with Covid-19 and four of the seven lungs from the patients with influenza, thrombi were consistently present in pulmonary arteries with a diameter of 1 mm to 2 mm, without complete luminal obstruction (Figs. S3 and S5). Fibrin thrombi of the alveolar capillaries could be seen in all the lungs from both groups of patients (Figure 2). Alveolar capillary microthrombi were 9 times as prevalent in patients with Covid-19 as in patients with influenza (mean [±SD] number of distinct thrombi per square centimeter of vascular lumen area, 159±73 and 16±16, respectively; P=0.002). Intravascular thrombi in postcapillary venules of less than 1 mm diameter were seen in lower numbers in the lungs from patients with Covid-19 than in those from patients with influenza (12±14 vs. 35±16, P=0.02). Two lungs in the Covid-19 group had involvement of all segments of the vasculature, as compared with four of the lungs in the influenza group; in three of the lungs in the Covid-19 group and three of the lungs in the influenza group, combined capillary and venous thrombi were found without arterial thrombi.
The histologic findings were supported by three-dimensional microCT of the pulmonary specimens: the lungs from patients with Covid-19 and from patients with influenza showed nearly total occlusions of precapillary and postcapillary vessels.
Figure 3. Microvascular Alterations in Lungs from Patients Who Died from Covid-19
We examined the microvascular architecture of the lungs from patients with Covid-19, lungs from patients with influenza, and uninfected control lungs with the use of scanning electron microscopy and microvascular corrosion casting. The lungs in the Covid-19 group had a distorted vascularity with structurally deformed capillaries (Figure 3). Elongated capillaries in the lungs from patients with Covid-19 showed sudden changes in caliber and the presence of intussusceptive pillars within the capillaries (Figure 3C). Transmission electron microscopy of the Covid-19 endothelium showed ultrastructural damage to the endothelium, as well as the presence of intracellular SARS-CoV-2 (Figure 3D). The virus could also be identified in the extracellular space.
Figure 4.Numeric Density of Features of Intussusceptive and Sprouting Angiogenesis in Lungs from Patients Who Died from Covid-19 or Influenza A(H1N1).
In the lungs from patients with Covid-19, the density of intussusceptive angiogenic features (mean [±SE], 60.7±11.8 features per field) was significantly higher than that in lungs from patients with influenza (22.5±6.9) or in uninfected control lungs (2.1±0.6) (P<0.001 for both comparisons) (Figure 4A). The density of features of conventional sprouting angiogenesis was also higher in the Covid-19 group than in the influenza group (Figure 4B). When the pulmonary angiogenic feature count was plotted as a function of the length of hospital stay, the degree of intussusceptive angiogenesis was found to increase significantly with increasing duration of hospitalization (P<0.001) (Figure 4C). In contrast, the lungs from patients with influenza had less intussusceptive angiogenesis and no increase over time (Figure 4C). A similar pattern was seen for sprouting angiogenesis (Figure 4D).Figure 5.elative Expression Analysis of Angiogenesis-Associated Genes in Lungs from Patients Who Died from Covid-19 or Influenza A(H1N1).
A multiplexed analysis of angiogenesis-related gene expression examining 323 genes from the nCounter PanCancer Progression Panel (NanoString Technologies) revealed differences between the specimens from patients with Covid-19 and those from patients with influenza. A total of 69 angiogenesis-related genes were differentially regulated only in the Covid-19 group, as compared with 26 genes differentially regulated only in the influenza group; 45 genes had shared changes in expression (Figure 5).
In this study, we examined the morphologic and molecular features of seven lungs obtained during autopsy from patients who died from SARS-CoV-2 infection. The lungs from these patients were compared with those obtained during autopsy from patients who had died from ARDS secondary to influenza A(H1N1) infection and from uninfected controls. The lungs from the patients with Covid-19 and the patients with influenza shared a common morphologic pattern of diffuse alveolar damage and infiltrating perivascular lymphocytes. There were three distinctive angiocentric features of Covid-19. The first feature was severe endothelial injury associated with intracellular SARS-CoV-2 virus and disrupted endothelial cell membranes. Second, the lungs from patients with Covid-19 had widespread vascular thrombosis with microangiopathy and occlusion of alveolar capillaries.12,19 Third, the lungs from patients with Covid-19 had significant new vessel growth through a mechanism of intussusceptive angiogenesis. Although our sample was small, the vascular features we identified are consistent with the presence of distinctive pulmonary vascular pathobiologic features in some cases of Covid-19.
Our finding of enhanced intussusceptive angiogenesis in the lungs from patients with Covid-19 as compared with the lungs from patients with influenza was unexpected. New vessel growth can occur by conventional sprouting or intussusceptive (nonsprouting) angiogenesis. The characteristic feature of intussusceptive angiogenesis is the presence of a pillar or post spanning the lumen of the vessel.20 Typically referred to as an intussusceptive pillar, this endothelial-lined intravascular structure is not seen by light microscopy but is readily identifiable by corrosion casting and scanning electron microscopy.21 Although tissue hypoxia was probably a common feature in the lungs from both these groups of patients, we speculate that the greater degree of endothelialitis and thrombosis in the lungs from patients with Covid-19 may contribute to the relative frequency of sprouting and intussusceptive angiogenesis observed in these patients. The relationship of these findings to the clinical course of Covid-19 requires further research to elucidate.
A major limitation of our study is that the sample was small; we studied only 7 patients among the more than 320,000 people who have died from Covid-19, and the autopsy data also represent static information. On the basis of the available data, we cannot reconstruct the timing of death in the context of an evolving disease process. Moreover, there could be other factors that account for the differences we observed between patients with Covid-19 and those with influenza. For example, none of the patients in our study who died from Covid-19 had been treated with standard mechanical ventilation, whereas five of the seven patients who died from influenza had received pressure-controlled ventilation. Similarly, it is possible that differences in detectable intussusceptive angiogenesis could be due to the different time courses of Covid-19 and influenza. These and other unknown factors must be considered when evaluating our data.22 Nonetheless, our analysis suggests that this possibility is unlikely, particularly since the degree of intussusceptive angiogenesis in the patients with Covid-19 increased significantly with increasing length of hospitalization, whereas in the patients with influenza it remained stable at a significantly lower level. Moreover, we have shown intussusceptive angiogenesis to be the predominant angiogenic mechanism even in late stages of chronic lung injury.21
ACE2 is an integral membrane protein that appears to be the host-cell receptor for SARS-CoV-2.23,24 Our data showed significantly greater numbers of ACE2-positive cells in the lungs from patients with Covid-19 and from patients with influenza than in those from uninfected controls. We found greater numbers of ACE2-positive endothelial cells and significant changes in endothelial morphology, a finding consistent with a central role of endothelial cells in the vascular phase of Covid-19. Endothelial cells in the specimens from patients with Covid-19 showed disruption of intercellular junctions, cell swelling, and a loss of contact with the basal membrane. The presence of SARS-CoV-2 virus within the endothelial cells, a finding consistent with other studies,25 suggests that direct viral effects as well as perivascular inflammation may contribute to the endothelial injury.
We report the presence of pulmonary intussusceptive angiogenesis and other pulmonary vascular features in the lungs of seven patients who died from Covid-19. Additional work is needed to relate our findings to the clinical course in these patients. To aid others in their research, our full data set is available on the Vivli platform (https://vivli.org/. opens in new tab) and can be requested with the use of the following digital object identifier: https://doi.org/10.25934/00005576. opens in new tab.
Supported by grants (HL94567 and HL134229, to Drs. Ackermann and Mentzer) from the National Institutes of Health, a grant from the Botnar Research Centre for Child Health (to Dr. Tzankov), a European Research Council Consolidator Grant (XHale) (771883, to Dr. Jonigk), and a grant (KFO311, to Dr. Jonigk) from Deutsche Forschungsgemeinschaft (Project Z2).
Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.
This article was published on May 21, 2020, at NEJM.org.
We thank Kerstin Bahr, Jan Hinrich Braesen, Peter Braubach, Emily Brouwer, Annette Mueller Brechlin, Regina Engelhardt, Jasmin Haslbauer, Anne Hoefer, Nicole Kroenke, Thomas Menter, Mahtab Taleb Naghsh, Christina Petzold, Vincent Schmidt, and Pauline Tittmann for technical support; Peter Boor of the German Covid-19 registry; and Lynnette Sholl, Hans Kreipe, Hans Michael Kvasnicka, and Jean Connors for helpful comments. Dr. Jonigk thanks Anita Swiatlak for her continued support.
From the Institute of Pathology and Department of Molecular Pathology, Helios University Clinic Wuppertal, University of Witten–Herdecke, Wuppertal (M.A.), the Institute of Functional and Clinical Anatomy, University Medical Center of the Johannes Gutenberg University Mainz, Mainz (M.A.), the Institute of Pathology (M.K., F.L., C.W., H.S., D.J.), the Department of Cardiothoracic, Transplantation, and Vascular Surgery (A.H.), and the Clinic of Pneumology (T.W.), Hannover Medical School, and the German Center for Lung Research, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH) (M.K., A.H., T.W., F.L., C.W., H.S., D.J.), Hannover — all in Germany; the Laboratory of Respiratory Diseases, BREATH, Department of Chronic Diseases, Metabolism, and Aging, KU Leuven, Leuven, Belgium (S.E.V., A.V.); the Institute of Pathology and Medical Genetics, University Hospital Basel, Basel, Switzerland (A.T.); and the Angiogenesis Foundation, Cambridge (W.W.L., V.W.L.), and the Laboratory of Adaptive and Regenerative Biology and the Division of Thoracic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston (S.J.M.) — all in Massachusetts.
Address reprint requests to Dr. Mentzer at the Division of Thoracic Surgery, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115, or at email@example.com.
Thromboembolism is a life-threatening manifestation of coronavirus disease 2019 (COVID-19). We investigated a dysfunctional phenotype of vascular endothelial cells in the lungs during COVID-19.
We obtained the lung specimens from the patients who died of COVID-19. The phenotype of endothelial cells and immune cells was examined by flow cytometry and immunohistochemistry (IHC) analysis. We tested the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endothelium using IHC and electron microscopy.
The autopsy lungs of COVID-19 patients exhibited severe coagulation abnormalities, immune cell infiltration, and platelet activation. Pulmonary endothelial cells of COVID-19 patients showed increased expression of procoagulant von Willebrand factor (VWF) and decreased expression of anticoagulants thrombomodulin and endothelial protein C receptor (EPCR). In the autopsy lungs of COVID-19 patients, the number of macrophages, monocytes, and T cells was increased, showing an activated phenotype. Despite increased immune cells, adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and P-selectin were downregulated in pulmonary endothelial cells of COVID-19 patients. Notably, decreased thrombomodulin expression in endothelial cells was associated with increased immune cell infiltration in the COVID-19 patient lungs. There were no SARS-CoV-2 particles detected in the lung endothelium of COVID-19 patients despite their dysfunctional phenotype. Meanwhile, the autopsy lungs of COVID-19 patients showed SARS-CoV-2 virions in damaged alveolar epithelium and evidence of hypoxic injury.
Pulmonary endothelial cells become dysfunctional during COVID-19, showing a loss of thrombomodulin expression related to severe thrombosis and infiltration, and endothelial cell dysfunction might be caused by a pathologic condition in COVID-19 patient lungs rather than a direct infection with SARS-CoV-2.
This work was supported by the Johns Hopkins University, the American Heart Association, and the National Institutes of Health.
Normally functioning endothelial cells protect the blood vessel by preventing unwanted coagulation. In COVID-19 patients, plasma levels of soluble markers associated with endothelial cell activation and dysfunction are elevated. To understand the phenotypic changes of vascular endothelial cells during COVID-19, we searched the PubMed database with the terms “COVID-19 endothelial cells” in combination with “phenotype”, “flow cytometry”, or “immunohistochemistry”. However, there was no article showing a comprehensive profile of endothelial cell phenotype in COVID-19.
Added value of this study
In this study, we report a phenotypic profile of endothelial cells in the COVID-19 patient lungs by using flow cytometry and immunohistochemistry (IHC) analysis. Pulmonary endothelial cells of COVID-19 patients exhibited a dysfunctional phenotype related to thrombosis. Especially, we found a loss of thrombomodulin expression, a potent anticoagulant and anti-inflammatory molecule, in endothelial cells of COVID-19 patients. As a mechanism of endothelial dysfunction in COVID-19, we tested if SARS-CoV-2 can directly infect endothelial cells. We found no clear evidence of SARS-CoV-2 infection in endothelial cells using IHC stain, electron microscopy, and in vitro viral infection.
Implications of all the available evidence
These findings provide evidence that endothelial cells may be a critical player to contribute to severe thrombosis in the lungs of COVID-19 patients and that endothelial thrombomodulin expression would be a potential therapeutic target for COVID-19-related immunothrombosis.
The global pandemic of coronavirus disease 2019 (COVID-19) is caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that primarily affects the respiratory tract.1 These respiratory effects can range from mild upper respiratory disease to severe pneumonia and acute respiratory distress syndrome (ARDS).1 However, patients hospitalized with COVID-19 also experienced an increased incidence of venous and arterial thromboembolic complications including acute pulmonary embolism (PE), deep vein thrombosis (DVT), ischemic stroke, or myocardial infarction.2, 3, 4 The incidence of thrombotic complications in intensive care unit patients has been reported between 16 and 49 percent.2, 3, 4 In a meta-analysis of 277 autopsy cases, small vessel thrombi were found in 10·8 percent of cases, and 19·1 percent had macrovascular thrombi such as DVT, PE, and mural thrombi.5 Patients hospitalized with COVID-19 have elevated levels of D-dimer, a thrombosis biomarker, and fibrin degradation products.6 Given the prominence of microthrombi in COVID-19, it was proposed to rename COVID-19 as MicroCLOTS (microvascular COVID-19 lung vessels obstructive thromboinflammatory syndrome).7Normally functioning vascular endothelium protects blood vessels by regulating vascular tone and preventing unwanted coagulation, playing an essential role in preventing ARDS.8,9 Endothelial cells express key molecules that regulate the balance between hemostasis and thrombosis such as thrombomodulin and von Willebrand factor (VWF).9, 10, 11, 12 In COVID-19 patients, elevated plasma levels of soluble VWF, thrombomodulin, intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), and P-selectin have been shown, suggesting endothelial cell activation and dysfunction.13, 14, 15, 16 Several researchers predicted that vascular endothelial cells in COVID-19 increased adhesion molecule expression for platelet and leukocyte migration and pro-inflammatory cytokine and chemokine production.8,17,18 Moreover, endothelial cell activation and damage were proposed to play a central role in the pathogenesis of COVID-19 associated with ARDS, vascular leakage, pulmonary edema, and a procoagulant state.8,18,19 Endothelial cell dysfunction was also expected to cause children’s multisystem inflammatory syndrome, which develops in a small percentage of children infected with SARS-CoV-2.20However, the phenotypic and transcriptional changes of endothelial cells in COVID-19 have not been fully described. Many single-cell RNA sequencing (scRNA-seq) studies on COVID-19 were performed with non-tissue samples such as peripheral blood mononuclear cells, bronchoalveolar lavage fluid, nasopharyngeal swab, and sputum, barely containing endothelial cells.21, 22, 23, 24, 25 Several studies using the lung tissues from human or non-human primates have been conducted, but they intended to investigate a host entry for SARS-CoV-2 – angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) – not showing a comprehensive profile of endothelial cell phenotype and transcriptome.26, 27, 28, 29, 30 Furthermore, endothelial transmembrane molecules such as thrombomodulin and ICAM-1 are generally cleaved by enzymes and released into the blood in diseased conditions, suggesting that a transcriptional profile of endothelial cells would not reflect their dysfunctional phenotype during COVID-19.11,12,31 Also, it remains unclear whether endothelial cell dysfunction and damage in COVID-19 are induced by direct SARS-CoV-2 infection or indirectly through pathologic conditions such as hypoxia and pro-inflammatory cytokine milieu. Some studies have shown ACE2 expression on endothelial cells with limited evidence for the direct infection by SARS-CoV-2.32,33 However, others have disputed ACE2 expression on endothelial cells and further infection by SARS-CoV-2.34, 35, 36, 37, 38, 39, 40, 41Here, we provide a unique perspective on the phenotypic changes of endothelial cells in the autopsy lungs, hearts, and kidneys of COVID-19 patients using flow cytometry and immunohistochemistry (IHC) analyses. Pulmonary endothelial cells showed a prothrombotic and dysfunctional phenotype during COVID-19, especially a loss of thrombomodulin expression, an anticoagulant and anti-inflammatory molecule. In addition, macrophages, monocytes, and T cells were increased and activated in the lungs of COVID-19 patients. However, we found no evidence of SARS-CoV-2 infection in the pulmonary endothelium, while the alveolar epithelium exhibited severe SARS-CoV-2 infection and damage, causing hypoxia during COVD-19. We confirmed hypoxic stress on endothelial cells and immune cells in the COVID-19 patient lungs.
We obtained seven lung samples, seven heart samples, and eight kidney samples from nine autopsies of patients who died of COVID-19 at the Johns Hopkins University (Table 1 and Supplementary Table S1). We were unable to collect all three tissues from all patients for technical reasons. As a non-COVID-19 control, eight lung specimens, eleven heart specimens, and ten kidney specimens were obtained from autopsies of patients who had no respiratory, cardiovascular, and renal diseases. Samples were fixed for histology study or cryopreserved for flow cytometry and RT-PCR analysis. For cryopreservation, samples were stabilized in HypoThermosol FRS preservation media (Stemcell) and then frozen in CryoStor CS10 cryopreservation media (Stemcell) at -80°C.Table 1Clinical and demographic characteristics of patients.
Written informed consent was obtained from subjects, and the study protocol was approved by the Committee for the Protection of Human Subjects (IRB NA_00036610). This study was conducted in accordance with the guidelines in the Declaration of Helsinki.
SARS-CoV-2 in vitro infection of primary human endothelial cells
Human coronary artery endothelial cells (HCAECs) were purchased from Cell Applications (300-05a). Cells were cultured in Human Meso Endo Growth Medium (Cell Applications). All infections were performed in a biosafety level (BSL) 3 environment. HCAECs were seeded onto 6-well plates at a density of 200,000 cells/well. At the time of infection, the growth medium was replaced with media containing SARS-CoV-2 (SARS-CoV-2/USA/DC-HP00007/2020, GISAID EPI_ISL_434688) at a multiplicity of infection (MOI) of 5 infectious units per cell.42 Cells were then incubated for 1 hour at 37°C. Media containing the virus was aspirated and replenished with fresh media. Samples were then incubated for 24 hours. Cell images were taken using an EVOS XL Core Imaging System (Invitrogen).
Lung, heart, and kidney tissues were paraffin-embedded, cut as 4-μm-thick sections, and stained with H&E. Coagulation abnormalities were graded by microscopic assessment of the severity of thrombus formation (graded from 0 to 5) and hemorrhage (from 0 to 5), and the sum of two item scores was shown. Infiltration was separately scored from 0 to 5. Grading was performed by two independent blinded investigators. Images were acquired on a BX43 microscope (Olympus) with a DS-Fi3 camera (Nikon) using NIS-Elements D Software (Nikon).
The tissue slides were deparaffinized, rehydrated, and blocked with Duel Endogenous Enzyme Block (Dako). Tissue sections were probed with the primary antibody against fibrin (59D8; Millipore), CD42b (SP219; Abcam; RRID: AB_2814749), VWF (F8/86; Invitrogen; RRID: AB_11001165), PECAM-1 (JC/70A; Abcam; RRID: AB_307284), or SARS-CoV-2 spike S1 protein (007; Sino Biological; AB_2827979) and then treated with HRP-conjugated secondary goat anti-rabbit or anti-mouse IgG antibody (Leica). The slides were counterstained with 50% hematoxylin in water.
Cell isolation from autopsy tissue samples
All procedures involving SARS-CoV-2-infected tissue samples were performed in a BSL3 environment until the virus is inactivated. Tissues were minced using a single-edge scalpel and then incubated with 5 mL of tissue digestion enzyme solution for 30 minutes at 37°C with intermittent vortexing. The digestion enzyme solution was prepared with 200 U/ml Collagenase (Worthington) for lungs or 400 U/ml for hearts and kidneys and supplemented with 50 U/ml DNase I (Worthington) and 50 U/ml Hyaluronidase (Sigma) for all organs. Cells were washed and filtered through 40 μm cell strainers (Falcon).
Flow cytometry samples from autopsy tissues were prepared in a BSL3 condition. Single cells were first stained with Live/Dead Fixable Aqua (Invitrogen) and 7-AAD (BioLegend). After blocking with TruStain FcX (BioLegend; RRID: AB_2818986) and normal mouse serum (Invitrogen), cells were surface-stained with fluorochrome-conjugated antibodies (Supplementary Table S2). Cells were then fixed, permeabilized with Cyto-Fast Fix/Perm Buffer (BioLegend), and stained with antibodies against intracellular antigens (Supplementary Table S2). Sample acquisition was performed on a BD FACSymphony flow cytometer (BD) running FACSDiva (BD). Results were analysed using FlowJo software (BD).
Total RNA from tissues or cultured cells was extracted using RNeasy Plus Mini Kit (Qiagen) or TRIzol (Invitrogen) in a BSL3 environment. Single‐stranded cDNA was synthesized with iScript Reverse Transcription Supermix (Bio-Rad). Target genes were amplified using Power SYBR Green PCR Master Mix (Applied Biosystems), and real-time cycle thresholds were detected via MyiQ2 thermal cycler (Bio-Rad). Data were analysed by the 2−ΔΔCt method and were normalized to GAPDH or HPRT expression and then to biological controls.
SARS-CoV-2 RNA detection
Total RNA extracted from autopsy tissues or cultured cells were reverse transcribed and then amplified with primers specific for SARS-CoV-2 nucleocapsid (N) genes. Primers for SARS-CoV-2 N1, SARS-CoV-2 N2, and internal control RNase P were obtained from the Integrated DNA Technologies. According to the Centers for Disease Control and Prevention (CDC) guideline, a test is considered positive for SARS-CoV-2 if both SARS-CoV-2 N1 and N2 markers show Ct value <40.
The lung tissue was excised from patient autopsies and cut into 2–3 mm3 pieces, and immediately fixed in 2·5% glutaraldehyde (EM grade; Electron Microscopy Sciences) dissolved in 0·1 M Na cacodylate, pH 7·4, for 2 hours at room temperature. Samples were processed as previously described by the Electron Microscopy Laboratory in the Department of Pathology at the Johns Hopkins University School of Medicine before examination with an electron microscope CM120 (Philips) under 80 kV.43 Images were acquired with Image Capture Engine V602 (Advanced Microscopy Techniques).
Wild-type male C57BL/6 mice (JAX 000664) were purchased from the Jackson Laboratory. Mice were housed in specific pathogen-free animal facilities at the Johns Hopkins University School of Medicine. Experiments were conducted with 6- to 10-week-old age-matched mice. For hypoxia exposure, mice were housed in Plexiglas hypoxia chambers with 10·0% O2 that had continuous airflow.44 The O2 concentration was monitored and controlled with a ProOx model 350 unit (BioSpherix) by infusion of N2. CO2 and ammonia were removed throughout the experiment. Control mice were exposed to normal room air (20·8% O2). On day 8 of hypoxia exposure, lungs were perfused with PBS and then harvested. All methods and protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University.
GraphPad Prism 8 software was used for statistical analysis. Data shown are mean values of two or three independent experiments. Data were analysed using non-parametric Mann-Whitney test or Kruskal-Wallis test followed by post-hoc Dunn’s multiple comparison test. Statistically significant comparisons were represented by asterisks in figures: *P < 0·05; **P < 0·005; ***P < 0·0005. Actual P-values were reported in figure legends. Linear regression using Spearman correlation was tested for correlation analysis. Actual r- and P-values were reported in figures.
Role of the funding source
The funding sources had no involvement in study design, data collection, data analyses, interpretation, or writing of this report.
Coagulation abnormality and infiltration are presented in the lungs of COVID-19 patients
Thromboembolism is the most common pathologic finding in the postmortem lungs of patients who died of COVID-19.45, 46, 47, 48 Thrombus formation is also observed in the autopsy hearts and kidneys from COVID-19 patients.46,48,49 To test whether thrombosis is present in the lungs, hearts, and kidneys from COVID-19 patient autopsies collected at the Johns Hopkins University, we analysed histologic sections of those tissue specimens. Clinical characteristics of our COVID-19 patient cohort are summarized in Table 1 and Supplementary Table S1. We tested seven lung samples, seven heart samples, and eight kidney samples collected from the autopsy of COVID-19 patients. Many postmortem lungs from COVID-19 patients showed severe coagulation abnormalities such as vascular congestion, thrombus formation, and hemorrhage, which were not observed in non-COVID-19 autopsy controls (Fig. 1a and Supplementary Fig. S1a). Fibrin deposition normally occurs at the site of the damaged blood vessel during coagulation, however, excessive formation or impaired clearance of a fibrin clot contributes to thrombosis development in pathologic conditions and diseases.50 IHC studies confirmed the development of fibrin- and platelet-rich thrombi in the lungs of COVID-19 patients (Fig. 1b and c and Supplementary Fig. S1b and c). The autopsy hearts and kidneys of COVID-19 patients exhibited congested blood vessels and fibrin-rich thrombi (Fig. 1a and b and Supplementary Fig. S1a and b). However, the severity of coagulation abnormalities in the hearts and kidneys of COVID-19 patients was milder compared to the lungs (Fig. 1d). No platelet-rich thrombi were found in the hearts and kidneys of COVID-19 patient autopsies (data not shown). Elevated plasma D-dimer levels confirmed thrombosis development in our COVID-19 patient cohort tested upon their hospitalization (Fig. 1e). In addition, we found that the number of platelets in the COVID-19 patient lungs was increased compared with controls by using flow cytometry (Fig. 1f and Supplementary Fig. S2). The number of platelets in the COVID-19 patient lungs was correlated with the severity of hypercoagulability (Fig. 1g). In the hearts and kidneys, the platelet counts were comparable between COVID-19 and control groups (Fig. 1f).
Severe infiltration was another histological finding in the lungs of COVID-19 patients (Fig. 1a). The number of CD45+ infiltrating cells was significantly increased in the COVID-19 patient lungs compared with controls (Fig. 1h and Supplementary Fig. S2). The number of CD45+ cells in the COVID-19 patient lungs analysed by flow cytometry was correlated with the infiltration score histologically graded (Fig. 1i). In contrast, CD45+ cell number was reduced in the hearts of COVID-19 patients compared to controls (Fig. 1h). The kidneys of COVID-19 patients presented a comparable number of CD45+ infiltrating cells to the controls (Fig. 1h). These results indicate that severe hypercoagulability and infiltration develop in the lungs of COVID-19 patients.
Platelets and endothelial cells show a prothrombotic phenotype in COVID-19 patient lungs
To investigate if platelets are activated in the lungs of COVID-19 patients, causing a severe hypercoagulable state, we characterized the phenotype of platelets in the autopsy lungs, hearts, and kidneys. In COVID-19 patients, blood platelets showed upregulated P-selectin and other receptors contributing to aggregation and activation of platelets.51,52 Our flow cytometry analysis revealed that platelet P-selectin level was increased in the hearts but not in the lungs and kidneys from COVID-19 patient autopsies compared to controls (Fig. 2a and b). We also tested the platelet expression of PF4 (CXCL4), which promotes blood coagulation and is a target of autoantibodies causing heparin-induced thrombocytopenia.53 Platelet PF4 expression showed a significant increase in the lungs of COVID-19 patients compared to controls (Fig. 2c and d). PF4 level in the heart platelets was also increased in the COVID-19 group, although it was not significant (Fig. 2c and d). In the kidneys, no different PF4 level was found between groups (Fig. 2c and d). Platelet expression of other receptors such as CD40L, CD42b (glycoprotein Ibα), and CXCR4 in the COVID-19 autopsy tissues was comparable to controls (data not shown). In addition, we found a massive production of VWF, a procoagulant, in most COVID-19 patient autopsy tissues – lungs, hearts, and kidneys – using IHC staining (Fig. 2e and Supplementary Fig. S3). VWF was detected in the outer layer of the thrombus and CD31-expressing vascular endothelium in the COVID-19 patient tissues, suggesting VWF expression by both activated platelets and endothelial cells. In contrast, control tissues showed a low level of VWF production only in the vascular endothelial lining (Fig. 2e and Supplementary Fig. S3).
Platelets exhibited an activated phenotype in the lungs, hearts, and kidneys of COVID-19 patients (Fig. 2a-e). However, severe hypercoagulability mainly developed in the lungs of our samples and barely in the hearts and kidneys (Fig. 1a-g). To test whether endothelial cells provide a prothrombotic environment specifically in the lungs during COVID-19, we examined a phenotype of endothelial cells in the lungs, hearts, and kidneys of COVID-19 patients using flow cytometry analysis. We found that endothelial cell expression of anticoagulants thrombomodulin and endothelial cell protein C receptor (EPCR) were significantly downregulated in the lung of COVID-19 patients compared with controls (Fig. 2f-h). Endothelial cells in the hearts of COVID-19 patients showed a slight decrease in thrombomodulin and EPCR expression, but it was not significant (Fig. 2f-h). The kidney endothelial cells expressed thrombomodulin and EPCR with no differences between groups (Fig. 2f-h). In hemostatic conditions, endothelial cells produce nitric oxide and prostaglandin I2 by utilizing the enzymes endothelial nitric oxide synthase (eNOS) and cyclooxygenase 2 (COX-2), respectively, to inhibit platelet aggregation and activation9. In the COVID-19 patient organs that we examined, endothelial cells expressed a comparable level of those enzymes to controls (data not shown). Collectively, endothelial cells represent a prothrombotic phenotype in COVID-19 patient lungs, leading to platelet recruitment and coagulation abnormalities.
Macrophages, monocytes, and T cells are increased and activated in COVID-19 patient lungs
We found an increased CD45+ cell infiltration in the lungs of COVID-19 patients (Fig. 1a and h). To further investigate the immune profile of COVID-19 patient lungs, we analysed the number and phenotype of different immune cells in the autopsy lungs of COVID-19 patients using flow cytometry. Macrophages were the largest immune cell population in both COVID-19 and control lungs and significantly increased in the COVID-19 patients compared with controls (Fig. 3a and Supplementary Fig. S4a). Similarly, the number of monocytes, CD4+ T cells, and CD8+ T cells was increased in the lungs of COVID-19 patients, while the neutrophil count was comparable between COVID-19 and controls (Fig. 3a and Supplementary Fig. S4a). In the phenotype analysis, macrophages in the COVID-19 patient lungs exhibited downregulated HLA-DR expression compared to controls (Fig. 3b and c). CD40 expression on macrophages was increased in the COVID-19 patients, although it was not significant (Fig. 3b and d). Monocytes in the COVID-19 patient lungs revealed upregulated CD16 expression compared with controls but not significantly (Fig. 3e and f). In CD4+ T cells of the COVID-19 patient lungs, CCR7+CD45RA− central memory T cells (TCM) were proportionally increased compared to controls, while CCR7−CD45RA+ effector memory T cells re-expressing CD45RA (TEMRA) were decreased (Fig. 3g and h). However, the absolute number of most CD4+ T cell subsets including CCR7+CD45+ naïve T cells (TN), TCM cells, and CCR7−CD45RA− effector memory T cells (TEM) was significantly increased in the COVID-19 patient lungs compared to controls (Fig. 3i). Similarly, the number of CD4+ TEMRA cells was inclined in the COVID-19 patient, although there was no significance (Fig. 3i). The cell frequencies and numbers of CD8+ T cell subsets in the COVID-19 patient lungs exhibited similar results to CD4+ T cells (Supplementary Fig. S4b and c). These data demonstrate that immune cells such as macrophages, monocytes, and T cells are increased and activated in the lungs during COVID-19.
Adhesion molecule expression is reduced in endothelial cells of COVID-19 patient lungs
We and others found increased immune cell infiltration in the autopsy lungs of COVID-19 patients (Fig. 3).45,48 Several researchers predicted that endothelial cell expression of adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and P-selectin would be upregulated to recruit immune cells in COVID-19.8,17,18 We tested whether adhesion molecule expression on endothelial cells is altered in the postmortem lungs, hearts, and kidneys from COVID-19 patients. Unexpectedly, endothelial adhesion molecule expression was reduced in the COVID-19 patient lungs compared to controls (Fig. 4a-f). In the lungs, endothelial cells in both COVID-19 and control groups were positive for ICAM-1 (Fig. 4a and b). However, we found a significant downregulation of ICAM-1 in the COVID-19 lungs. Endothelial cells in the control lungs expressed a low level of VCAM-1, E-selectin, and P-selectin, while the COVID-19 lungs showed even lower expression of those molecules (Fig. 4a, c, d, e, and f). In the COVID-19 autopsy hearts, ICAM-1 and E-selectin were significantly downregulated compared to controls, while VCAM-1 and P-selectin expression levels showed no differences (Fig. 4a-f). Endothelial cells in the kidneys from COVID-19 patients expressed a comparable level of adhesion molecules to controls except VCAM-1 (Fig. 4a-f). VCAM-1 was significantly decreased in the COVID-19 kidneys (Fig. 4a and c).
In the COVID-19 patient lungs, a loss of ICAM-1 expression in endothelial cells was not associated with the severity of immune cell infiltration, suggesting that other mechanisms would be involved in the pulmonary infiltration (Fig. 4g). Decreased expression of thrombomodulin in endothelial cells was significantly correlated with an increased infiltration in the lungs of COVID-19 patients (Fig. 4h). Thrombomodulin plays an anti-inflammatory role as well as an anticoagulant function.11,12,54,55 These data suggest that endothelial cell dysfunction with downregulated adhesion molecules occurs in the lungs of COVID-19 patients and that downregulated thrombomodulin expression in endothelial cells is related to severe infiltration during COVID-19.
SARS-CoV-2 does not directly infect endothelial cells or induce their dysfunction
To understand if SARS-CoV-2 directly infects endothelial cells leading to a prothrombotic and dysfunctional state, we first tested the presence of the SARS-CoV-2 genome in the lungs, hearts, and kidneys from COVID-19 patients. All COVID-19 lung specimens were positive for viral RNA in the RT-PCR test (Fig. 5a and Supplementary Fig. S5a). However, two of seven heart samples and six of eight kidney samples tested positive for SARS-CoV-2 RNA (Fig. 5a and Supplementary Fig. S5b and c). Second, we searched SARS-CoV-2 spike protein in the COVID-19 autopsy tissues using IHC stain to detect virus-infected cells. The spike protein was present in all SARS-CoV-2 RNA-positive tissue specimens and was located outside the vascular endothelial layer in the lungs, hearts, and kidneys of COVID-19 patients (Fig. 5b and Supplementary Fig. S5d). Significantly less viral antigen was detected in the heart and kidney tissues compared to the lungs. We could not detect SARS-CoV-2 virions in the vascular endothelium of COVID-19 patient lungs using electron microscopy but were able to identify virus-like particles in the epithelium (Fig. 5c). Those particles represented a SARS-CoV-2-specific ultrastructure described by Miller and Goldsmith.56 In addition, alveolar epithelial cells of the COVID-19 patients showed an abnormal ultrastructure, which has been observed in ciliary dyskinesia induced by respiratory syncytial virus (RSV) infection (Fig. 5c).57
Since endothelial cells of the COVID-19 patient lungs exhibited a prothrombotic and dysfunctional phenotype despite no signs of a direct SARS-CoV-2 infection, we hypothesized that endothelial cell dysfunction is not caused directly by the viral infection. To test this hypothesis, we infected HCAECs with SARS-CoV-2 in vitro. HCAECs showed no cytopathic changes at 16 hours post-infection (Fig. 5d). In the RT-PCR test, we found no detectable level of virus RNA in both SARS-CoV-2 and mock infection groups (Supplementary Fig. S5e). Importantly, no downregulation of thrombomodulin, ICAM-1, and VCAM-1 was shown in the SARS-CoV-2-infected HCAECs compared with mock infection control (Fig. 5e). Thus, we did not find evidence of direct endothelial cell infection by SARS-CoV-2 despite a dysfunctional phenotype of endothelial cells in COVID-19 patient lungs.
Epithelial cells are involved in endothelial dysfunction, coagulation, and infiltration in COVID-19 patient lungs
Nasal epithelial cells and type II alveolar epithelial cells in the respiratory system are a primary target of SARS-CoV-2 entry, and the resultant lung injury can cause hypoxia in COVID-19.58, 59, 60 Since we found the SARS-CoV-2 infection and ultrastructural damage in alveolar epithelial cells of COVID-19 patients (Fig. 5c), we tested whether a hypoxic injury occurred in the lungs. In our COVID-19 patient cohort, the average oxygen saturation before death was 76·8% and ranged between 49% and 100% (Fig. 6a, Table 1 and Supplementary Table S1). The mRNA level of HIF1A, a hypoxia-induced gene, was upregulated in the COVID-19 patient lungs compared to controls, while the hearts and kidneys showed no differences (Fig. 6b). In the COVID-19 patients, lung epithelial cells showed increased GLUT1 expression, another hypoxia-induced molecule, compared to controls, although epithelial HIF1α expression was comparable between COVID-19 and control groups (Fig. 6c). Endothelial cells and immune cells in the lungs of COVID-19 patients exhibited increased HIF1α and GLUT1 expression, confirming hypoxic stress in the lungs (Fig. 6d and Supplementary Fig. S6). To investigate if hypoxia can directly cause endothelial cell dysfunction in the lungs, we housed naïve mice in the hypoxia chamber with 10% O2 for eight days (Fig. 6e). In the hypoxia-exposed mice, the lung endothelial cells showed decreased thrombomodulin and ICAM-1 expression compared to the normal oxygen control, mimicking the dysfunctional phenotype of lung endothelial cells in the COVID-19 patients (Fig. 6f and Supplementary Fig. S7). Other adhesion molecules including VCAM-1, E-selectin, and P-selectin were expressed at comparable levels between the hypoxia and control groups (Fig. 6f and data not shown).
Next, we tested if damaged epithelial cells are directly involved in a hypercoagulable state and immune cell infiltration during COVID-19. We found that pulmonary epithelial cells in the COVID-19 patients expressed a higher level of tissue factor, a procoagulant, compared with controls (Fig. 6g and h). Elevated tissue factor expression level in epithelial cells was significantly associated with increased platelet number in the COVID-19 patient lungs (Fig. 6i). In addition, unlike endothelial cells, pulmonary epithelial cells exhibited an increase of ICAM-1 expression in the COVID-19 patients compared to controls, although it was not significant (Fig. 6g and h). These data suggest that pulmonary epithelial cells might participate in endothelial cell dysfunction, platelet activation and aggregation, and immune cell infiltration directly or indirectly via hypoxia during COVID-19.
Many researchers have speculated about a phenotype of activated endothelial cells in COVID-19.8,17,18 However, direct evidence was missing. Most human studies on SARS-CoV-2 infection have used the blood or pharyngeal swab specimens that can be conveniently acquired but barely contain endothelial cells.61 In this study, we elucidated the dysfunctional phenotype of endothelial cells associated with hypercoagulability and severe infiltration in the lungs during fatal COVID-19. As previously shown by other researchers, we found severe coagulation abnormalities and infiltration in the autopsy lungs from COVID-19 patients.45, 46, 47, 48 Pulmonary endothelial cells exhibited prothrombotic and dysfunctional phenotype with upregulated procoagulant and downregulated anticoagulants and adhesion molecules in COVID-19. However, we were unable to find clear evidence of direct endothelial cell infection by SARS-CoV-2 in the postmortem COVID-19 specimens and the primary cell in vitro culture. This suggests that endothelial cell dysfunction might not be directly caused by SARS-CoV-2 infection but rather induced by the pathophysiologic conditions during COVID-19. Unlike the lungs, the hearts and kidneys in COVID-19 showed a mild level of hypercoagulable state, infiltration, and endothelial cell dysfunction.Several researchers predicted activated and dysfunctional phenotype of endothelial cells showing strong expression of procoagulant VWF in COVID-19.17,18 Indeed, we found that VWF expression was strikingly increased in endothelial cells of the lungs, hearts, and kidneys from COVID-19 patients. The VWF is a multimeric glycoprotein secreted exclusively by endothelial cells and platelets into the circulation and involved in thrombus formation by tethering platelets to endothelial cells.9,10 In addition, we found the downregulation of anticoagulants thrombomodulin and EPCR in the lung endothelial cells of COVID-19 autopsies. Thrombomodulin is a transmembrane glycoprotein expressed mainly on endothelial cells but also found in immune cells, vascular smooth muscle cells, keratinocytes, and lung alveolar epithelial cells.9,11,12 Thrombomodulin is a potent anticoagulant that binds to thrombin and then deactivates it to initiate an anticoagulation cascade. Thrombin-thrombomodulin complex activates protein C, which is a crucial mediator to inactivate coagulant Factors Va and VIIIa and to further reduce thrombin generation in the anticoagulation processes.11,12,62 EPCR, another coagulant, presents the protein C to the thrombin-thrombomodulin complex to accelerate protein C activation.11,62 This suggests that endothelial cell dysfunction with increased procoagulant production and decreased anticoagulant expression is associated with severe thrombus formation and hypercoagulability in the lung of COVID-19 patients.In the hearts and kidneys of COVID-19 patients, platelets showed an activated phenotype with increased P-selectin, PF4, and VWF expression at a similar level as the lungs. Furthermore, endothelial cells largely produced VWF to a similar degree in the lungs, hearts, and kidneys in the COVID-19 patients. However, the hearts and kidneys from COVID-19 patients exhibited only mild coagulation abnormalities, while the lungs developed fatal thrombosis. This suggests that platelet activation and partial dysfunction of endothelial cells are insufficient for developing a severe multi-organ hypercoagulable state during COVID-19. A loss of thrombomodulin and EPCR expression on endothelial cells seems critical for the progression to fatal thrombotic disorders. Previous studies showed that endothelial expression of those anticoagulant molecules is downregulated in thrombosis associated with meningococcal sepsis, purpura fulminans, and cerebral malaria.63, 64, 65 In mice, endothelial cell-specific thrombomodulin deletion resulted in lethal thrombosis development.66 We found a significant loss of thrombomodulin and EPCR expression in the lungs of COVID-19 patients. In contrast, those anticoagulants in the hearts and kidneys were relatively preserved. Recently, decreased endothelial thrombomodulin and ECPR expression in COVID-19 patient lungs was reported by using IHC stain.67In COVID-19 patients, it is reported that the plasma level of soluble thrombomodulin was elevated.13,15,16 Since pathophysiological conditions such as infection, sepsis, inflammation, and ischemic disease can cause thrombomodulin release from the endothelial cell membrane into the blood, soluble thrombomodulin is considered as a useful biomarker to diagnose endothelial damage and dysfunction.11,12 Meanwhile, those disease conditions can downregulate endothelial thrombomodulin mRNA transcription through hypoxic stress and pro-inflammatory cytokine milieu.11 We found hypoxic stress in the endothelial cells of COVID-19 patient lungs. Thus, our finding of decreased thrombomodulin expression on endothelial cells in COVID-19 might be mediated by two regulation processes – release in a soluble form and downregulation of gene transcription.We elucidated an increase of macrophages, monocytes, CD4+ T cells, and CD8+ T cells in the postmortem lungs of COVID-19 patients using flow cytometry analysis, while other researchers have mainly utilized IHC or scRNA-seq analysis.68, 69, 70 In our analysis, lung macrophages of COVID-19 patients expressed a lower level of HLA-DR and a higher level of CD40 than those of controls. Severe COVID-19 patients showed an increased number of HLA-DRlo monocytes in the blood, a dysregulated or immunosuppressive subset of monocytes.25,71, 72, 73 This suggests that abnormally or alternatively activated macrophages emerged in the lungs during COVID-19. The role of CD40 expressed on macrophages in COVID-19 remains unclear. However, SARS-CoV-2 infection increased CD40 expression and decreased HLA-DR expression in M2 macrophages in vitro, supporting our findings on the lung macrophages of COVID-19 patients.74 Our data revealed an increase of CD16+ inflammatory monocytes in the autopsy lungs of COVID-19 patients. Some studies showed an elevated number of CD14−CD16+ non-classical monocytes in COVID-19 patient blood, while others disagreed.25,71,75,76 Additionally, we showed that the number of CD4+ TEM and CD8+ TEM cells was significantly increased in the COVID-19 patient lungs. In agreement with this, other studies showed an elevated frequency or number of activated T cells in the blood during COVID-19, although they have used different markers to define T cell subsets than ours.77,78In SARS-CoV-2 infection, it is speculated that vascular endothelial cells exhibit increased expression of adhesion molecules.8,17,18 ICAM-1 upregulation is generally mediated by various biological stimuli such as inflammation, bacteria or virus infection, and oxidative stress.79 Endothelial cell expression of other adhesion molecules including VCAM-1, E-selectin, and P-selectin would be increased similarly to ICAM-1 upregulation. In pathologic conditions, adhesion molecules anchored in endothelial cell transmembrane are cleaved by matrix metalloproteinases (MMPs) and released into the blood.31 Several studies showed that soluble forms of ICAM-1, VCAM-1, E-selectin, and P-selectin are highly elevated in severely ill COVID-19 patients.13,14 In our flow cytometry analysis, the lung endothelial cells in COVID-19 exhibited reduced expression of surface ICAM-1, VCAM-1, E-selectin, and P-selectin despite increased pulmonary infiltration. It is unclear why adhesion molecule expression was decreased in activated endothelial cells in COVID-19. Since CD45+ cell number was increased in the lungs of COVID-19 patients, it could be hypothesized that endothelial cells upregulate adhesion molecules upon SARS-CoV-2 infection to recruit immune cells from the blood, and then the adhesion molecule expression is downregulated after the infiltration. As a possible cause of downregulation, activated endothelial cells release endothelial microparticles (EMPs), which induce ICAM-1 downregulation on adjacent endothelial cells.80 In addition, COVID-19 patients showed excessively increased plasma MMP level and enzymatic activity, suggesting the massive cleavage of membrane-bound adhesion molecules on endothelial cells.81, 82, 83 More studies will be necessary to determine how ICAM-1 and other adhesion molecules are downregulated on endothelial cells after the immune cell infiltration in COVID-19. Meanwhile, we found that decreased endothelial thrombomodulin expression was significantly associated with increased infiltration in the lungs of COVID-19 patients, while ICAM-1 expression showed no correlation. Thrombomodulin plays an anti-inflammatory role besides its anticoagulant function by inhibiting immune cell adhesion and suppressing complement system activation.11,12,54,55 Thus, a loss of thrombomodulin expression on endothelial cells could trigger or accelerate a pathogenic infiltration of immune cells into the lungs during COVID-19.Since we observed prothrombotic endothelial cells with downregulated adhesion molecule expression in the COVID-19 patient lungs, we wanted to examine the cause of endothelial cell dysfunction. We investigated the hypothesis that SARS-CoV-2 directly infects endothelial cells leading to dysfunction. Although we detected SARS-CoV-2 RNA by RT-PCR test in all autopsy lung specimens with COVID-19, the spike protein was not seen in endothelial cells by IHC stain. We found the SARS-CoV-2 particles in the pulmonary epithelium but not in the endothelium using electron microscopy. Interestingly, the SARS-CoV-2 virions were mostly located around the ciliated epithelium, where highly expresses ACE2 and TMPRSS2 allowing a strong SARS-CoV-2 infection.22 In addition, we were unable to infect endothelial cells in vitro with SARS-CoV-2 or induce endothelial cell dysfunction by the virus infection. Thus, endothelial cells are unlikely to be infected directly by SARS-CoV-2. Several recent studies showed the low susceptibility of endothelial cells to SARS-CoV-2 due to low ACE2 expression.35, 36, 37, 38, 39, 40, 41 This is supported by the fact that recombinant ACE2 transduction is required for sufficient SARS-CoV-2 infection of endothelial cells.40 Some researchers published electron microscope studies showing SARS-CoV-2 particles present in endothelial cells.33,47 However, others clarified that those shown particles were indeed cellular ribosomal complexes or vesicles.34,84We sought a pathophysiological condition in SARS-CoV-2 infection that can mediate endothelial cell dysfunction. We found a low oxygen saturation in the COVID-19 patients of our cohort and an increased HIF1A gene expression in their lungs, supporting hypoxic stress during COVID-19. The protein levels of HIF1α or GLUT1 were upregulated in epithelial cells, endothelial cells, and immune cells of COVID-19 patient lungs, confirming a hypoxic condition in SARS-CoV-2-infected lungs. Hypoxia can cause endothelial cell dysfunction and further thrombosis.85,86 In our mouse experiment, hypoxia induced endothelial cell dysfunction exhibiting thrombomodulin and ICAM-1 downregulation, which we observed in the COVID-19 patient lungs. Thus, pulmonary epithelial cells infected and damaged by SARS-CoV-2 could cause hypoxia leading to endothelial dysfunction in COVID-19 patients. Wang et al. reported that epithelial cells were more susceptible to SARS-CoV-2 infection compared with endothelial cells in their in vitro co-culture system.38 Co-cultured endothelial cells in the system revealed altered proteomic profile despite no direct SARS-CoV-2 infection, suggesting crosstalk between SARS-CoV-2-infected epithelial cells and endothelial cells. In addition, we found an increased expression of tissue factor and ICAM-1 in pulmonary epithelial cells of COVID-19 patients. Thus, injured epithelial cells may be directly involved in thrombosis and infiltration in the lungs of COVID-19 patients. Alternatively, microparticles derived from platelets, monocytes, and endothelial cells can act as a procoagulant contributing to thrombosis development.87 Elevated circulating microparticles have been reported in COVID-19 patients, suggesting a pathogenic role of microparticles in COVID-19-related immunothrombosis.88 Future studies should explore whether microparticles are involved in thrombosis development and further endothelial cell dysfunction in the lungs of COVID-19 patients.Microbial infection can be a common cause of inflammation-involved thrombosis.89 Notably, one-half of COVID-19 non-survivors revealed a secondary infection diagnosed by clinical symptoms, signs, or culture tests in Wuhan.90 In our COVID-19 patient cohort, only two revealed a suspected secondary infection tested by sputum culture, suggesting thrombosis mainly caused by primary SARS-CoV-2 infection or following pathologic conditions.In this study, we demonstrated the downregulation of endothelial thrombomodulin in the COVID-19 autopsies and suggested a prominent role of thrombomodulin in preventing severe thrombosis and infiltration. In murine models, overexpression of human thrombomodulin or the engineered preservation of thrombomodulin expression protected the lungs from thrombotic disorders and inflammation.54,91 Moreover, recombinant thrombomodulin or thrombomodulin analog Solulin treatment ameliorated the infarct volume in ischemic stroke.92,93 Recombinant soluble thrombomodulin administration was also beneficial in patients with disseminated intravascular coagulation and different inflammatory diseases.12,55,94 Based on our finding of endothelial thrombomodulin downregulation in COVID-19, recombinant thrombomodulin treatment could be considered as a potential therapy.In conclusion, we found a procoagulant phenotype of endothelial cells with upregulated VWF and downregulated thrombomodulin and EPCR in the lungs and, to a lesser extent, hearts and kidneys during COVID-19. The number of macrophages, monocytes, and T cells was increased in the lungs of COVID-19 patients, and all of them exhibited activated phenotype. Despite increased infiltration, endothelial cell expression of ICAM-1, VCAM-1, E-selectin, and P-selectin was downregulated in the lungs of COVID-19 patients. However, decreased thrombomodulin expression in endothelial cells was related to increased infiltration in the COVID-19 patient lungs. We showed that endothelial cell dysfunction was not caused directly by SARS-CoV-2 infection. Interestingly, we found pulmonary epithelial cell infection and damage by SARS-CoV-2 in the COVID-19 patients. Hypoxia caused by infected epithelial cells would lead to endothelial cell dysfunction in COVID-19. The main limitation of our study is the small sample size. In addition, since we did not have access to biopsy samples of COVID-19 patients, our study was conducted using autopsy specimens. Further studies confirming our findings in fresh biopsy samples from COVID-19 patients are needed.
T.W. and D.C. conceptualized study. J.S., C.R.P., A.Z.R., and J.E.H. collected autopsy samples. T.W., M.K.W., D.M.H., M.V.T., Z.M., J.T.S., B.A., I.C., and A.P. performed experiments and analysed data. I.C., A.P., and D.C. verified the underlying data. A.P. supervised BSL3 experiments. T.W. and D.C. wrote the manuscript. E.G., M.K.H., A.G.H., D.-H.K., I.C., R.A.J., N.A.G., J.E.H., A.P., and D.C. reviewed the manuscript and provided intellectual input. All authors reviewed and approved the final version of the manuscript. All authors had full access to all the data in this study and accepted the responsibility to submit for publication.
Declaration of interests
This work was supported by the Johns Hopkins COVID-19 Research Response Program to D.C. and C.R.P., the COVID-19 Rapid Response Grant 814664 from the American Heart Association (AHA) to D.C., the National Institutes of Health (NIH) Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C to A.P., the NIH/National Heart, Lung, and Blood Institute (NHLBI) R01HL122606 and NIH/National Institute of General Medicine Sciences (NIGMS) R01GM110674 to R.A.J., and the NIH/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Supplemental Award R01DK093770-09S1 for COVID-19 Research to C.R.P. D.C. is supported by the NIH/NHLBI R01HL118183 and R01HL136586, and AHA 20TPA35490421 and 19TPA34910007. D.C. and N.A.G. are supported by the Matthew Vernon Poyner Memorial Foundation. J.E.H. received support from NIH/National Cancer Institute (NCI) P30CA006973, Cancer Clinical Care support grant and P50CA06924, supplement to a GI SPORE. N.A.G. is supported by the AHA 20SFRN35380046 and NIH/NHLBI R01HL118183. D.-H.K is supported by the NIH/National Institute of Allergy and Infectious Diseases (NIAID) R01AI143773. A.G.H. is supported by NIH/NHLBI R01HL147660. T.W. is supported by the 2018 Rhett Lundy Memorial Research Fellowship from the Myocarditis Foundation. M.K.W. is funded by the NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) F31AR077406. D.M.H. is funded by the NIH/NHLBI F31HL149328. The funding sources had no involvement in study design, data collection, analyses, interpretation, or writing of this report.
Data sharing statement
All data reported in this article and supplementary materials are available for sharing by requesting to the corresponding author.
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Figure 1Hypercoagulable state and infiltration in lungs during COVID-19. (a) Representative images of H&E staining on postmortem lungs, hearts, and kidneys from COVID-19 patients and non-COVID-19 controls. Scale bars: 25 µm. (b) Representative images of immunohistochemistry (IHC) for fibrin (brown) on autopsy lungs, hearts, and kidneys of COVID-19 patients. Scale bars: 25 µm. (c) Representative image of IHC for CD42b (brown) on autopsy lungs of COVID-19 patients. Scale bar: 25 µm. (d) Coagulation abnormality score of COVID-19 patient lungs, hearts, and kidneys examined by H&E stain. Kruskal-Wallis test with Dunn’s multiple comparisons test was used for statistical analysis. P-values: 0·0260 (Lung vs Heart); 0·0201 (Lung vs Kidney); 0·9999 (Heart vs Kidney). (e) Plasma D-dimer level in COVID-19 patients upon their hospitalization. (f) Number of platelets in postmortem lungs, hearts, and kidneys from COVID-19 patients and controls determined by flow cytometry. Platelets were gated on EpCAM−CD45−PECAM-1dimCD41+ (Supplementary Fig. S2). Mann-Whitney test was used for statistical analysis. P-values: 0·0205 (Lung); 0·3845 (Heart); 0·9999 (Kidney). (g) Correlation between platelet number and coagulation abnormality score in autopsy lungs of COVID-19 patients. Spearman correlation was used for statistical analysis. (h) Number of CD45+ cells in COVID-19 patient lungs, hearts, and kidneys. CD45+ immune cells were gated on EpCAM−CD45+ (Supplementary Fig. S2). Mann-Whitney test was used for statistical analysis. P-values: 0·0037 (Lung); 0·0122 (Heart); 0·8286 (Kidney). (i) Correlation between CD45+ infiltrating cell number examined by flow cytometry and infiltration score histologically graded in postmortem lungs of COVID-19 patients. Spearman correlation was used for statistical analysis. In COVID-19 patient group, seven lung samples, seven heart samples, and eight kidney samples were tested. In controls, eight lung samples, eleven heart samples, and ten kidney samples were used. Data shown are mean values of two or three independent experiments. *P < 0·05; **P < 0·005.
Figure 2Prothrombotic phenotype of platelets and endothelial cells in COVID-19 patient lungs. (a and b) Flow cytometry analysis (a) and frequencies (b) of P-selectin expression on platelets in lungs, hearts, and kidneys of COVID-19 patients and controls. Platelets were gated on EpCAM−CD45−PECAM-1dimCD41+ (Supplementary Fig. S2). Mann-Whitney test was used for statistical analysis. P-values: 0·2319 (Lung); 0·0003 (Heart); 0·0676 (Kidney). (c and d) Histograms (c) and mean fluorescent intensity (MFI) (d) of PF4 expression in platelets of lungs, hearts, and kidneys from COVID-19 patients and controls. Mann-Whitney test was used for statistical analysis. P-values: 0·0289 (Lung); 0·1422 (Heart); 0·7577 (Kidney). (e) Representative images of immunohistochemistry staining for VWF (red) and CD31 (brown) on lung, heart, and kidney sections from COVID-19 and control autopsies. Scale bars: 12·5 µm. (f) Histograms for thrombomodulin (TM) and endothelial protein C receptor (EPCR) expression on endothelial cells in postmortem lungs, hearts, and kidneys. Endothelial cells were gated on EpCAM−CD45−PECAM-1hiCD34+ (Supplementary Fig. S2). (g and h) MFI of endothelial TM (g) and EPCR (h) expression in lungs, hearts, and kidneys from COVID-19 and control autopsies. Mann-Whitney test was used for statistical analysis. P-values: 0·0022 (Lung); 0·2109 (Heart); 0·1728 (Kidney) (g). P-values: 0·0003 (Lung); 0·0556 (Heart); 0·5884 (Kidney) (h). In COVID-19 patient group, seven lung samples, seven heart samples, and eight kidney samples were tested. In controls, eight lung samples, eleven heart samples, and ten kidney samples were used. Data shown are mean values of two or three independent experiments. *P < 0·05; **P < 0·005, ***P < 0·0005.
Figure 3Increased macrophages, monocytes, and T cells with activated phenotype in lungs of COVID-19 patients. (a) Numbers of macrophages (CD68+CD206+), monocytes (CD68+CD206−CD169−), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), and neutrophils (CD15+CD66b+) in autopsy lungs of COVID-19 patients and controls. The gating strategy for flow cytometry analysis was shown in Supplementary Fig. S4a. Mann-Whitney test was used for statistical analysis. P-values: 0·0041 (Macrophages); 0·0140 (Monocytes); 0·0205 (CD4+ T cells); 0·0093 (CD8+ T cells); 0·4634 (Neutrophils). (b) Histograms of macrophage HLA-DR and CD40 expression in COVID-19 patient lungs. (c and d) Mean fluorescent intensity (MFI) of HLA-DR expression (c) and frequency of CD40 expression (d) in lung macrophages from COVID-19 and control autopsies. Mann-Whitney test was used for statistical analysis. P-values: 0·0175 (c); 0·0541 (d). (e and f) Flow cytometry analysis (e) and frequency (f) of CD16 expression in monocytes of autopsy lungs from COVID-19 patients. Mann-Whitney test was used for statistical analysis. P-value: 0·1807. (g) CCR7 and CD45RA expression profiles of CD4+ T cells from COVID-19 and control lungs. (h and i) Frequencies of T cell subsets among CD4+ T cells (h) and absolute numbers (i) in postmortem lungs of COVID-19 patients and controls. Mann-Whitney test was used for statistical analysis. P-values: 0·6943 (CCR7+CD45RA+); 0·0289 (CCR7+CD45RA−); 0·6126 (CCR7−CD45RA−); 0·0037 (CCR7−CD45RA+) (h). P-values: 0·0037 (CCR7+CD45RA+); 0·0022 (CCR7+CD45RA−); 0·0205 (CCR7−CD45RA−); 0·0721 (CCR7−CD45RA+) (i). Seven COVID-19 lung samples and eight control lung samples were tested. Data shown are mean values of two or three independent experiments. *P < 0·05; **P < 0·005.
Figure 4Downregulated adhesion molecules in lung endothelial cells during COVID-19. (a) Flow cytometry analysis of ICAM-1 and VCAM-1 expression on endothelial cells of postmortem lungs, hearts, and kidneys from COVID-19 patients and controls. (b) Mean fluorescent intensity (MFI) of ICAM-1 expression on endothelial cells in lungs, hearts, and kidneys from COVID-19 and control autopsies. Mann-Whitney test was used for statistical analysis. P-values: 0·0037 (Lung); 0·0154 (Heart); 0·2031 (Kidney). (c) Frequencies of VCAM-1+ cells among endothelial cells in postmortem lungs, hearts, and kidney specimens. Mann-Whitney test was used for statistical analysis. P-values: 0·0401 (Lung); 0·2463 (Heart); 0·0263 (Kidney). (d) Flow cytometry plots of E-selectin and P-selectin expression on endothelial cells of autopsy lungs, hearts, and kidneys from COVID-19 patients and controls. (e and f) MFI of E-selectin (e) and P-selectin (f) expression in endothelial cells from postmortem lungs, hearts, and kidneys. Mann-Whitney test was used for statistical analysis. P-values: 0·0059 (Lung); 0·0268 (Heart); 0·5726 (Kidney) (e). P-values: 0·0939 (Lung); 0·0853 (Heart); 0·8968 (Kidney) (f). (g and h) Correlation between endothelial ICAM-1 (g) or thrombomodulin (TM) (h) expression and infiltration score histologically evaluated. Spearman correlation was used for statistical analysis. In COVID-19 patient group, seven lung samples, seven heart samples, and eight kidney samples were tested. In controls, eight lung samples, eleven heart samples, and ten kidney samples were used. Data shown are mean values of two or three independent experiments. *P < 0·05; **P < 0·005.
Figure 5No endothelial cell infection or dysfunction by SARS-CoV-2. (a) Pie charts showing the presence of SARS-CoV-2 RNA tested by RT-PCR in postmortem lungs, hearts, and kidneys from COVID-19 patients. Each pie slice represents one patient. (b) Representative images of immunohistochemistry for SARS-CoV-2 spike protein (brown) on lungs, hearts, and kidneys from COVID-19 autopsies. Scale bars: 25 µm (top); 6·25 µm (bottom). Arrows indicate the blood vessel. (c) Representative images of SARS-CoV-2 in the pulmonary epithelium of COVID-19 patient. Scale bars: 2 µm (left); 200 nm (right). (d) Microscopic images of primary human coronary artery endothelial cells (HCAECs) with in vitro SARS-CoV-2 or mock infection. Images were acquired 16 hours after virus inoculation. Scale bars: 125 µm. (e) Frequencies of thrombomodulin (TM)+, ICAM-1+, and VCAM-1+ cells among HCAECs with in vitro SARS-CoV-2 or mock infection assessed by flow cytometry. Mann-Whitney test was used for statistical analysis. P-values: 0·7000 (TM); 0·9999 (ICAM-1); 0·9999 (VCAM-1). Seven lung samples, seven heart samples, and eight kidney samples were tested. Data shown are mean values of two or three independent experiments.
Julia Hippisley-Cox1, Duncan Young2,3, Carol Coupland4, Keith M Channon5, Pui San Tan6, David A Harrison7, Kathryn Rowan8, Paul Aveyard6, Ian D Pavord9, Peter J Watkinson5,10
Correspondence to Prof Julia Hippisley-Cox, Primary Care Health Sciences, University of Oxford, Oxford OX1
There is uncertainty about the associations of angiotensive enzyme (ACE) inhibitor and angiotensin receptor blocker (ARB) drugs with COVID-19 disease. We studied whether patients prescribed these drugs had altered risks of contracting severe COVID-19 disease and receiving associated intensive care unit (ICU) admission.
This was a prospective cohort study using routinely collected data from 1205 general practices in England with 8.28 million participants aged 20–99 years. We used Cox proportional hazards models to derive adjusted HRs for exposure to ACE inhibitor and ARB drugs adjusted for sociodemographic factors, concurrent medications and geographical region. The primary outcomes were: (a) COVID-19 RT-PCR diagnosed disease and (b) COVID-19 disease resulting in ICU care.
Of 19 486 patients who had COVID-19 disease, 1286 received ICU care. ACE inhibitors were associated with a significantly reduced risk of COVID-19 disease (adjusted HR 0.71, 95% CI 0.67 to 0.74) but no increased risk of ICU care (adjusted HR 0.89, 95% CI 0.75 to 1.06) after adjusting for a wide range of confounders. Adjusted HRs for ARBs were 0.63 (95% CI 0.59 to 0.67) for COVID-19 disease and 1.02 (95% CI 0.83 to 1.25) for ICU care.
There were significant interactions between ethnicity and ACE inhibitors and ARBs for COVID-19 disease. The risk of COVID-19 disease associated with ACE inhibitors was higher in Caribbean (adjusted HR 1.05, 95% CI 0.87 to 1.28) and Black African (adjusted HR 1.31, 95% CI 1.08 to 1.59) groups than the white group (adjusted HR 0.66, 95% CI 0.63 to 0.70). A higher risk of COVID-19 with ARBs was seen for Black African (adjusted HR 1.24, 95% CI 0.99 to 1.58) than the white (adjusted HR 0.56, 95% CI 0.52 to 0.62) group.
ACE inhibitors and ARBs are associated with reduced risks of COVID-19 disease after adjusting for a wide range of variables. Neither ACE inhibitors nor ARBs are associated with significantly increased risks of receiving ICU care. Variations between different ethnic groups raise the possibility of ethnic-specific effects of ACE inhibitors/ARBs on COVID-19 disease susceptibility and severity which deserves further study.
It might sound strange, but the respiratory system and the digestive system depend on one another for optimal function. Because oxygen is essential to the proper functioning of the body, one of the main concerns for people with chronic lung diseases is maintaining enough oxygen in their blood. The body needs energy and oxygen, so let’s take a closer look at oxygen levels and the digestive system.
What does the digestive system do?
The digestive system breaks down food so that it can become energy for the body. The digestive system is comprised of a complex system of organs, nerves, hormones, bacteria and blood work together to digest food. Digestive organs include the stomach, small intestines, large intestines, liver, pancreas and gall bladder.
What’s the connection between the respiratory system, oxygen levels and the digestive system?
The respiratory and digestive systems work together to power the body. A properly functioning respiratory system delivers adequate oxygen to the blood. Because the digestive system breaks down food and uses muscular contractions to move food through the digestive tract, it needs oxygen to function properly.
In turn, the respiratory system depends on a properly functioning digestive system to provide the fuel it needs to work effectively. Each function of the body depends on other functions, and all parts of the body need fuel and oxygen.
What are the risks of having lung disease and digestive system conditions?
In many cases, oxygen levels and the digestive system go hand-in-hand. COPD and other chronic lung diseases carry a risk for certain digestive disorders. Because some foods and drinks can cause symptom flare-ups, it’s important to know what to eat and what to avoid. Foods such as dairy and cruciferous vegetables are linked to increased mucus production and gas. Certain foods can also make GERD symptoms worse.
GERD or gastroesophageal reflux disease is common among people with COPD. GERD is a digestive disorder in which the stomach valve that keeps stomach acid down weakens or malfunctions, allowing stomach acid into the esophagus. If stomach acid reaches the lungs, it can result in irritation, increased coughing and shortness of breath.
GERD Symptoms include:
Hoarseness or sore throat
Burning in the chest or throat
Sensation of a lump in the throat
Regurgitation of stomach contents
What can I do to improve my blood oxygen levels?
Talk with your doctor about any new or worsening symptoms. See your doctor regularly, even if you’re feeling well. Now that you have information about oxygen levels and the digestive system, discuss your oxygen, food and exercise needs with your doctor. You and your physician can decide, together, on the best treatment plan for you.
Cellular therapy also helps many people with chronic lung diseases breathe easier by promoting the healing of lung tissue from within the body. The Lung Health Institute extracts cells from a patient’s blood separates them and then returns them intravenously. The cells may travel with the blood through the heart and into the lungs to become oxygenated. Once in the lungs, the majority of the cells become trapped in the pulmonary trap, and the now oxygen-rich blood travels to the rest of the body. In fact, many patients report improved lung function and are able to come off their supplemental oxygen after treatment.
On rounds in a 20-bed intensive care unit one recent day, physician Joshua Denson assessed two patients with seizures, many with respiratory failure and others whose kidneys were on a dangerous downhill slide. Days earlier, his rounds had been interrupted as his team tried, and failed, to resuscitate a young woman whose heart had stopped. All shared one thing, says Denson, a pulmonary and critical care physician at the Tulane University School of Medicine. “They are all COVID positive.”
As the number of confirmed cases of COVID-19 surges past 2.2 million globally and deaths surpass 150,000, clinicians and pathologists are struggling to understand the damage wrought by the coronavirus as it tears through the body. They are realizing that although the lungs are ground zero, its reach can extend to many organs including the heart and blood vessels, kidneys, gut, and brain.
“[The disease] can attack almost anything in the body with devastating consequences,” says cardiologist Harlan Krumholz of Yale University and Yale-New Haven Hospital, who is leading multiple efforts to gather clinical data on COVID-19. “Its ferocity is breathtaking and humbling.”
Understanding the rampage could help the doctors on the front lines treat the fraction of infected people who become desperately and sometimes mysteriously ill. Does a dangerous, newly observed tendency to blood clotting transform some mild cases into life-threatening emergencies? Is an overzealous immune response behind the worst cases, suggesting treatment with immune-suppressing drugs could help? What explains the startlingly low blood oxygen that some physicians are reporting in patients who nonetheless are not gasping for breath? “Taking a systems approach may be beneficial as we start thinking about therapies,” says Nilam Mangalmurti, a pulmonary intensivist at the Hospital of the University of Pennsylvania (HUP).
What follows is a snapshot of the fast-evolving understanding of how the virus attacks cells around the body, especially in the roughly 5% of patients who become critically ill. Despite the more than 1000 papers now spilling into journals and onto preprint servers every week, a clear picture is elusive, as the virus acts like no pathogen humanity has ever seen. Without larger, prospective controlled studies that are only now being launched, scientists must pull information from small studies and case reports, often published at warp speed and not yet peer reviewed. “We need to keep a very open mind as this phenomenon goes forward,” says Nancy Reau, a liver transplant physician who has been treating COVID-19 patients at Rush University Medical Center. “We are still learning.”
The infection begins
When an infected person expels virus-laden droplets and someone else inhales them, the novel coronavirus, called SARS-CoV-2, enters the nose and throat. It finds a welcome home in the lining of the nose, according to a preprint from scientists at the Wellcome Sanger Institute and elsewhere. They found that cells there are rich in a cell-surface receptor called angiotensin-converting enzyme 2 (ACE2). Throughout the body, the presence of ACE2, which normally helps regulate blood pressure, marks tissues vulnerable to infection, because the virus requires that receptor to enter a cell. Once inside, the virus hijacks the cell’s machinery, making myriad copies of itself and invading new cells.
As the virus multiplies, an infected person may shed copious amounts of it, especially during the first week or so. Symptoms may be absent at this point. Or the virus’ new victim may develop a fever, dry cough, sore throat, loss of smell and taste, or head and body aches.
If the immune system doesn’t beat back SARS-CoV-2 during this initial phase, the virus then marches down the windpipe to attack the lungs, where it can turn deadly. The thinner, distant branches of the lung’s respiratory tree end in tiny air sacs called alveoli, each lined by a single layer of cells that are also rich in ACE2 receptors.
Normally, oxygen crosses the alveoli into the capillaries, tiny blood vessels that lie beside the air sacs; the oxygen is then carried to the rest of the body. But as the immune system wars with the invader, the battle itself disrupts this healthy oxygen transfer. Front-line white blood cells release inflammatory molecules called chemokines, which in turn summon more immune cells that target and kill virus-infected cells, leaving a stew of fluid and dead cells—pus—behind. This is the underlying pathology of pneumonia, with its corresponding symptoms: coughing; fever; and rapid, shallow respiration (see graphic). Some COVID-19 patients recover, sometimes with no more support than oxygen breathed in through nasal prongs.
But others deteriorate, often quite suddenly, developing a condition called acute respiratory distress syndrome (ARDS). Oxygen levels in their blood plummet and they struggle ever harder to breathe. On x-rays and computed tomography scans, their lungs are riddled with white opacities where black space—air—should be. Commonly, these patients end up on ventilators. Many die. Autopsies show their alveoli became stuffed with fluid, white blood cells, mucus, and the detritus of destroyed lung cells.
Authors: February 19, 2021Source:Taylor & Francis Group
A review of 57 studies shows people with asthma had a 14 percent lower risk of getting COVID-19 and were significantly less likely to be hospitalized with the virus.
A new study looking at how COVID-19 affects people with asthma provides reassurance that having the condition doesn’t increase the risk of severe illness or death from the virus.
George Institute for Global Health researchers in Australia analysed data from 57 studies with an overall sample size of 587,280. Almost 350,000 people in the pool had been infected with COVID-19 from Asia, Europe, and North and South America and found they had similar proportions of asthma to the general population.
The results, published in the peer-reviewed Journal of Asthma, show that just over seven in every 100 people who tested positive for COVID-19 also had asthma, compared to just over eight in 100 in the general population having the condition. They also showed that people with asthma had a 14 percent lower risk of acquiring COVID-19 and were significantly less likely to be hospitalized with the virus.
There was no apparent difference in the risk of death from COVID-19 in people with asthma compared to those without.
Head of The Institute’s Respiratory Program, co-author Professor Christine Jenkins said that while the reasons for these findings weren’t clear, there were some possible explanations — such as some inhalers perhaps limiting the virus’ ability to attach to the lungs.
“Chemical receptors in the lungs that the virus binds to are less active in people with a particular type of asthma and some studies suggest that inhaled corticosteroids — commonly used to treat asthma — can reduce their activity even further,” she said.
“Also, initial uncertainty about the impact of asthma on COVID-19 may have caused anxiety among patients and caregivers leading them to be more vigilant about preventing infection.”
Lead author Dr Anthony Sunjaya added that while this study provides some reassurance about the risks of exposure to COVID-19 in people with asthma, doctors and researchers were still learning about the effects of the virus.
“While we showed that people with asthma do not seem to have a higher risk of infection with COVID-19 compared to those without asthma and have similar outcomes, we need further research to better understand how the virus affects those with asthma,” he said.